Protease variants and polynucleotides encoding same

ABSTRACT

The present invention relates to protease variants and methods for obtaining protease variants. The present invention also relates to polynucleotides encoding the variants; nucleic acid constructs, vectors, and host cells comprising the polynucleotides; and methods of using the variants.

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form,which is incorporated herein by reference.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates to novel protease variants exhibitingalterations relative to the parent protease in one or more propertiesincluding: wash performance, detergent stability and/or storagestability. The variants of the invention are suitable for use incleaning processes and detergent compositions, such as laundrycompositions and dish wash compositions, including hand wash andautomatic dish wash compositions. The present invention also relates toisolated DNA sequences encoding the variants, expression vectors, hostcells, and methods for producing and using the protease variants of theinvention.

Description of the Related Art

Enzymes have been used within the detergent industry as part of washingformulations for many decades. Proteases are from a commercialperspective the most relevant enzyme in such formulations, but otherenzymes including lipases, amylases, cellulases, hemicellulases ormixtures of enzymes are also often used. To improve the cost and/or theperformance of proteases there is an ongoing search for proteases withaltered properties, such as increased activity at low temperatures,increased stability, increased specific activity at a given pH, alteredCa²⁺ dependency, increased stability in the presence of other detergentingredients (e.g. bleach, surfactants etc.) etc. One family ofproteases, which are widely used in detergents, are the subtilases. Thisfamily has previously been further grouped into 6 different sub-groupsby Siezen R J and Leunissen J A M, 1997, Protein Science, 6, 501-523.One of these sub-groups is the Subtilisin family which includessubtilases such as BPN′, subtilisin 309 (SAVINASE®, Novozymes A/S),subtilisin Carlsberg (ALCALASE®, Novozymes A/S), subtilisin S41 (asubtilase from the psychrophilic Antarctic Bacillus TA41, Davail S etal. 1994, The Journal of Biological Chemistry, 269(26), 99. 17448-17453)and subtilisin S39 (a subtilase from the psychrophilic AntarcticBacillus TA39, Narinx E et al. 1997, Protein Engineering, 10 (11), pp.1271-1279). The TY-145 protease is a subtilase from Bacillus sp. TY-145,NCIMB 40339, which was first described in WO 92/17577 (Novozymes A/S)and in the later application WO2004/067737 (Novozymes A/S) disclosingthe three-dimensional structure and the use of protein engineering toalter functionality of a TY-145 subtilase.

SUMMARY OF THE INVENTION

The invention further relates to protease variants comprises one or moresubstitutions selected from the group consisting of Q70F, Q70A, Q70N,S111R, S111E, S111D, S114A, S114Q, S144R, A145E, K146T, K146N, K146W,K146F, K146A, I150A, I150N, I150N, I150S,A151R, N176Y, I178Y, I178F,I178P, G182A, L184F, L184Y, L184F, L184Y, L184W, L184D, R224D, R224G,R224S, and Y240R, wherein the positions corresponds to the positions ofSEQ ID NO: 3, wherein the variant has a sequence identity to SEQ ID NO:3 of at least 70% and wherein the variant has protease activity.

The present invention also relates to isolated polynucleotides encodingthe variants; nucleic acid constructs, vectors, and host cellscomprising the polynucleotides; and methods of producing the variants.

Overview of Sequences Listing

SEQ ID NO: 1=is the DNA sequence of TY-145 protease isolated fromBacillus sp.

SEQ ID NO: 2=is the amino acid sequence as deduced from SEQ ID NO: 1.

SEQ ID NO: 3=is the amino acid sequence of the mature TY-145 protease.

SEQ ID NO: 4=is the amino acid sequence of theTY-145protease+S173P+S175P.

SEQ ID NO: 5=is the amino acid sequence of theTY TY-145protease+S173P+S175P+F180Y.

Definitions

The term “protease” is defined herein as an enzyme that hydrolysespeptide bonds. It includes any enzyme belonging to the EC 3.4 enzymegroup (including each of the thirteen subclasses thereofhttp://en.wikipedia.org/wiki/Category:EC_3.4). The EC number refers toEnzyme Nomenclature 1992 from NC-IUBMB, Academic Press, San Diego,Calif., including supplements 1-5 published in Eur. J. Biochem. 1994,223, 1-5; Eur. J. Biochem. 1995, 232, 1-6; Eur. J. Biochem. 1996, 237,1-5; Eur. J. Biochem. 1997, 250, 1-6; and Eur. J. Biochem. 1999, 264,610-650; respectively. The term “subtilases” refer to a sub-group ofserine protease according to Siezen et al., Protein Engng. 4 (1991)719-737 and Siezen et al. Protein Science 6 (1997) 501-523. Serineproteases or serine peptidases is a subgroup of proteases characterizedby having a serine in the active site, which forms a covalent adductwith the substrate. Further, the subtilases (and the serine proteases)are characterized by having two active site amino acid residues apartfrom the serine, namely a histidine and an aspartic acid residue. Thesubtilases may be divided into 6 sub-divisions, i.e. the Subtilisinfamily, the Thermitase family, the Proteinase K family, the Lantibioticpeptidase family, the Kexin family and the Pyrolysin family. The term“protease activity” means a proteolytic activity (EC 3.4). Proteases ofthe invention are endopeptidases (EC 3.4.21). For purposes of thepresent invention, protease activity is determined according to theprocedure described in “Materials and Methods” below. The proteasevariants of the present invention have at least 20%, e.g., at least 40%,at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 95%, or at least 100% of the protease activity of the polypeptidewith SEQ ID NO: 3.

The term “parent”, “protease parent” or “precursor protease” means aprotease to which an alteration is made to produce the enzyme variantsof the present invention. Thus the parent is a protease having theidentical amino acid sequence of said variant but not having thealterations at one or more of said specified positions. It will beunderstood, that in the present context the expression “having identicalamino acid sequence” relates to 100% sequence identity. The parent maybe a naturally occurring (wild-type) polypeptide. In a particularembodiment the parent is a protease with at least 60%, at least 61%, atleast 62%, at least 63%, at least 64%, at least 65%, at least 70%, atleast 72%, at least 73%, at least 74%, at least 75%, at least 76%, atleast 77%, at least 78%, at least 79%, at least 80%, at least 81%, atleast 82%, at least 83%, at least 84%, at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99% or 100% identity to a polypeptidewith SEQ ID NO: 3.

The term “protease variant” means a protease having protease activitycomprising an alteration, i.e., a substitution, insertion, and/ordeletion, preferably substitution, at one or more (or one or several)positions compared to its parent which is a protease having theidentical amino acid sequence of said variant but not having thealterations at one or more of said specified positions. A substitutionmeans a replacement of an amino acid occupying a position with adifferent amino acid; a deletion means removal of an amino acidoccupying a position; and an insertion means adding amino acids e.g. 1to 10 amino acids, preferably 1-3 amino acids adjacent to an amino acidoccupying a position. Preferably the variant is modified by the hand ofman. In one aspect, the variant is at least 1% pure, e.g., at least 5%pure, at least 10% pure, at least 20% pure, at least 40% pure, at least60% pure, at least 80% pure, and at least 90% pure, as determined by SDSPAGE.

The term “isolated polynucleotide” means a polynucleotide that ismodified by the hand of man. In one aspect, the isolated polynucleotideis at least 1% pure, e.g., at least 5% pure, at least 10% pure, at least20% pure, at least 40% pure, at least 60% pure, at least 80% pure, atleast 90% pure, and at least 95% pure, as determined by agaroseelectrophoresis. The polynucleotides may be of genomic, cDNA, RNA,semisynthetic, synthetic origin, or any combinations thereof.

The term “allelic variant” means any of two or more alternative forms ofa gene occupying the same chromosomal locus. Allelic variation arisesnaturally through mutation, and may result in polymorphism withinpopulations. Gene mutations can be silent (no change in the encodedpolypeptide) or may encode polypeptides having altered amino acidsequences. An allelic variant of a polypeptide is a polypeptide encodedby an allelic variant of a gene.

The term “substantially pure variant” means a preparation that containsat most 10%, at most 8%, at most 6%, at most 5%, at most 4%, at most 3%,at most 2%, at most 1%, and at most 0.5% by weight of other polypeptidematerial with which it is natively or recombinantly associated.Preferably, the variant is at least 92% pure, e.g., at least 94% pure,at least 95% pure, at least 96% pure, at least 97% pure, at least 98%pure, at least 99%, at least 99.5% pure, and 100% pure by weight of thetotal polypeptide material present in the preparation. The variants ofthe present invention are preferably in a substantially pure form. Thiscan be accomplished, for example, by preparing the variant by well-knownrecombinant methods or by classical purification methods.

The term “wild-type protease” means a protease expressed by a naturallyoccurring organism, such as a bacterium, archaea, yeast, fungus, plantor animal found in nature. An example of a wild-type protease is theTY-145 protease.

The term “mature polypeptide” means a polypeptide in its final formfollowing translation and any post-translational modifications, such asN-terminal processing, C-terminal truncation, glycosylation,phosphorylation, etc. In one aspect, the mature polypeptide correspondsto the amino acid sequence with SEQ ID NO: 3.

The term “mature polypeptide coding sequence” means a polynucleotidethat encodes a mature polypeptide having protease activity. In oneaspect, the mature polypeptide coding sequence is nucleotides 331 to1263 of SEQ ID NO: 1 based on the SignalP (Nielsen et al., 1997, ProteinEngineering 10: 1-6)] that predicts nucleotides 1 to 81 of SEQ ID NO: 1is the signal peptide.

The term “cDNA” means a DNA molecule that can be prepared by reversetranscription from a mature, spliced, mRNA molecule obtained from aprokaryotic or eukaryotic cell. A cDNA lacks intron sequences that maybe present in the corresponding genomic DNA. The initial, primary RNAtranscript is a precursor to mRNA that is processed through a series ofsteps, including splicing, before appearing as mature spliced mRNA.

The term “coding sequence” means a polynucleotide, which directlyspecifies the amino acid sequence of its polypeptide product. Theboundaries of the coding sequence are generally determined by an openreading frame, which usually begins with the ATG start codon oralternative start codons such as GTG and TTG and ends with a stop codonsuch as TAA, TAG, and TGA. The coding sequence may be a DNA, cDNA,synthetic, or recombinant polynucleotide.

The term “nucleic acid construct” means a nucleic acid molecule, eithersingle- or double-stranded, which is isolated from a naturally occurringgene or is modified to contain segments of nucleic acids in a mannerthat would not otherwise exist in nature or which is synthetic. The termnucleic acid construct is synonymous with the term “expression cassette”when the nucleic acid construct contains the control sequences requiredfor expression of a coding sequence of the present invention.

The term “operably linked” means a configuration in which a controlsequence is placed at an appropriate position relative to the codingsequence of a polynucleotide such that the control sequence directs theexpression of the coding sequence.

The term “control sequences” means all components necessary for theexpression of a polynucleotide encoding a variant of the presentinvention. Each control sequence may be native or foreign to thepolynucleotide encoding the variant or native or foreign to each other.Such control sequences include, but are not limited to, a leader,polyadenylation sequence, propeptide sequence, promoter, signal peptidesequence, and transcription terminator. At a minimum, the controlsequences include a promoter, and transcriptional and translational stopsignals. The control sequences may be provided with linkers for thepurpose of introducing specific restriction sites facilitating ligationof the control sequences with the coding region of the polynucleotideencoding a variant.

The term “expression” includes any step involved in the production ofthe variant including, but not limited to, transcription,post-transcriptional modification, translation, post-translationalmodification, and secretion.

The term “expression vector” means a linear or circular DNA moleculethat comprises a polynucleotide encoding a variant and is operablylinked to additional nucleotides that provide for its expression.

The term “transcription promoter” is used for a promoter which is aregion of DNA that facilitates the transcription of a particular gene.Transcription promoters are typically located near the genes theyregulate, on the same strand and upstream (towards the 5′ region of thesense strand).

The term “transcription terminator” is used for a section of the geneticsequence that marks the end of gene or operon on genomic DNA fortranscription.

The term “host cell” means any cell type that is susceptible totransformation, transfection, transduction, and the like with a nucleicacid construct or expression vector comprising a polynucleotide of thepresent invention. The term “host cell” encompasses any progeny of aparent cell that is not identical to the parent cell due to mutationsthat occur during replication.

The relatedness between two amino acid sequences or between twonucleotide sequences is described by the parameter “sequence identity”.For purposes of the present invention, the degree of sequence identitybetween two amino acid sequences is determined using theNeedleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol.48: 443-453) as implemented in the Needle program of the EMBOSS package(EMBOSS: The European Molecular Biology Open Software Suite, Rice etal., 2000, Trends Genet. 16: 276-277), preferably version 3.0.0 orlater. The optional parameters used are gap open penalty of 10, gapextension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62)substitution matrix. The output of Needle labeled “longest identity”(obtained using the −nobrief option) is used as the percent identity andis calculated as follows:

(Identical Residues×100)/(Length of Alignment−Total Number of Gaps inAlignment)

The term “high stringency conditions” means for probes of at least 100nucleotides in length, prehybridization and hybridization at 42° C. in5×SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon spermDNA, and 50% formamide, following standard Southern blotting proceduresfor 12 to 24 hours. The carrier material is finally washed three timeseach for 15 minutes using 2×SSC, 0.2% SDS at 65° C.

The term “very high stringency conditions” means for probes of at least100 nucleotides in length, prehybridization and hybridization at 42° C.in 5×SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmonsperm DNA, and 50% formamide, following standard Southern blottingprocedures for 12 to 24 hours. The carrier material is finally washedthree times each for 15 minutes using 2×SSC, 0.2% SDS at 70° C.

The term “medium stringency conditions” means for probes of at least 100nucleotides in length, prehybridization and hybridization at 42° C. in5×SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon spermDNA, and 35% formamide, following standard Southern blotting proceduresfor 12 to 24 hours. The carrier material is finally washed three timeseach for 15 minutes using 2×SSC, 0.2% SDS at 55° C.

The term “medium-high stringency conditions” means for probes of atleast 100 nucleotides in length, prehybridization and hybridization at42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml sheared and denaturedsalmon sperm DNA, and either 35% formamide, following standard Southernblotting procedures for 12 to 24 hours. The carrier material is finallywashed three times each for 15 minutes using 2×SSC, 0.2% SDS at 60° C.

The term “low stringency conditions” means for probes of at least 100nucleotides in length, prehybridization and hybridization at 42° C. in5×SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon spermDNA, and 25% formamide, following standard Southern blotting proceduresfor 12 to 24 hours. The carrier material is finally washed three timeseach for 15 minutes using 2×SSC, 0.2% SDS at 50° C.

The term “very low stringency conditions” means for probes of at least100 nucleotides in length, prehybridization and hybridization at 42° C.in 5×SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmonsperm DNA, and 25% formamide, following standard Southern blottingprocedures for 12 to 24 hours. The carrier material is finally washedthree times each for 15 minutes using 2×SSC, 0.2% SDS at 45° C.

The term “improved property” means a characteristic associated with avariant that is improved compared to the parent or compared to aprotease with SEQ ID NO: 3, or compared to a protease having theidentical amino acid sequence of said variant but not having thealterations at one or more of said specified positions. Such improvedproperties include, but are not limited to, wash performance, proteaseactivity, thermal activity profile, thermostability, pH activityprofile, pH stability, substrate/cofactor specificity, improved surfaceproperties, substrate specificity, product specificity, increasedstability, improved stability under storage conditions, and chemicalstability.

The term “improved protease activity” is defined herein as an alteredprotease activity (as defined above) e.g. by increased proteinconversion of a protease variant displaying an alteration of theactivity relative (or compared) to the activity of the parent protease,or compared to a protease with SEQ ID NO: 3, or relative to a proteasehaving the identical amino acid sequence of said variant but not havingthe alterations at one or more of said specified positions, by increasedprotein conversion.

The term “stability” includes storage stability and stability duringuse, e.g. during a wash process and reflects the stability of theprotease variant according to the invention as a function of time e.g.how much activity is retained when the protease variant is kept insolution in particular in a detergent solution. The stability isinfluenced by many factors e.g. pH, temperature, detergent compositione.g. amount of builder, surfactants etc. The terms “improved stability”and “increased stability” includes “improved chemical stability”,“detergent stability” or “improved detergent stability.

The term “improved chemical stability” is defined herein as a variantenzyme displaying retention of enzymatic activity after a period ofincubation in the presence of a chemical or chemicals, either naturallyoccurring or synthetic, which reduces the enzymatic activity of theparent enzyme. Improved chemical stability may also result in variantsbeing more able to catalyze a reaction in the presence of suchchemicals. The term “improved thermal activity” means a variantdisplaying an altered temperature-dependent activity profile at aspecific temperature relative to the temperature-dependent activityprofile of the parent or relative to a protease with SEQ ID NO: 3. Inone embodiment the protease variants according to the invention haveimproved inhibitor binding compared to the parent enzyme. In oneparticular embodiment the protease variants of the invention haveimproved inhibition by inhibitor compared to the parent e.g. compared toSEQ ID NO 3. In another particular embodiment the protease variants ofthe present invention have increased inhibition by a protease inhibitorcompared to the inhibition of the TY-145 protease (SEQ ID NO 3) by thesame inhibitor as described in Example 2 in Materials and Method herein.

An irreversible inhibitor causes covalent modification of the enzyme, sothat its activity is permanently reduced. The interaction between theprotease variants of the invention and an inhibitor suitable for theprotease variants of the invention are preferably reversible thus theeffect of the inhibitor is reversed by removing the inhibitor. Theinhibitor constant, Ki, is an indication of the potency of an inhibitorand it is the concentration required to obtain half of the maximuminhibition. The Ki is a measure of the effectiveness of the inhibitionof a specific function. In this case the effectiveness of inhibition orstabilizing the protease variants of the present invention. The proteasevariants of the invention are improved compared to the parent proteasesby being more inhibited or stabilized by a suitable inhibitor than thecorresponding parent protease therefore Ki is lower for the proteasevariants according to the invention than Ki for the correspondingparent.

The term “improved wash performance” is defined herein as a proteasevariant according to the invention displaying an improved washperformance relative to the wash performance of the parent protease,relative to a protease with SEQ ID NO: 3 or relative to a proteasehaving the identical amino acid sequence of said variant but not havingthe alterations at one or more of said specified positions when measuredin a relevant assay such as AMSA. The term “wash performance” includeswash performance in laundry but also e.g. in hand wash and dish wash.The wash performance may be quantified as described under the definitionof “improved wash performance” herein. The term “low temperatureperformance” is defined herein as a protease variant according to theinvention having wash performance as described above at or below 20° C.

The term “detergent composition”, includes unless otherwise indicated,granular or powder-form all-purpose or heavy-duty washing agents,especially cleaning detergents; liquid, gel or paste-form all-purposewashing agents, especially the so-called heavy-duty liquid (HDL) types;liquid fine-fabric detergents; hand dishwashing agents or light dutydishwashing agents, especially those of the high-foaming type; machinedishwashing agents, including the various tablet, granular, liquid andrinse-aid types for household and institutional use; liquid cleaning anddisinfecting agents, including antibacterial hand-wash types, cleaningbars, soap bars, mouthwashes, denture cleaners, car or carpet shampoos,bathroom cleaners; hair shampoos and hair-rinses; shower gels, foambaths; metal cleaners; as well as cleaning auxiliaries such as bleachadditives and “stain-stick” or pre-treat types. The terms “detergentcomposition” and “detergent formulation” are used in reference tomixtures which are intended for use in a wash medium for the cleaning ofsoiled objects. In some embodiments, the term is used in reference tolaundering fabrics and/or garments (e.g., “laundry detergents”). Inalternative embodiments, the term refers to other detergents, such asthose used to clean dishes, cutlery, etc. (e.g., “dishwashingdetergents”). It is not intended that the present invention be limitedto any particular detergent formulation or composition. The term“detergent composition” is not intended to be limited to compositionsthat contain surfactants. It is intended that in addition to thevariants according to the invention, the term encompasses detergentsthat may contain, e.g., surfactants, builders, chelators or chelatingagents, bleach system or bleach components, polymers, fabricconditioners, foam boosters, suds suppressors, dyes, perfume, tannishinhibitors, optical brighteners, bactericides, fungicides, soilsuspending agents, anti corrosion agents, enzyme inhibitors orstabilizers, enzyme activators, transferase(s), hydrolytic enzymes,oxido reductases, bluing agents and fluorescent dyes, antioxidants, andsolubilizers.

The term “fabric” encompasses any textile material. Thus, it is intendedthat the term encompass garments, as well as fabrics, yarns, fibers,non-woven materials, natural materials, synthetic materials, and anyother textile material.

The term “textile” refers to woven fabrics, as well as staple fibers andfilaments suitable for conversion to or use as yarns, woven, knit, andnon-woven fabrics. The term encompasses yarns made from natural, as wellas synthetic (e.g., manufactured) fibers. The term, “textile materials”is a general term for fibers, yarn intermediates, yarn, fabrics, andproducts made from fabrics (e.g., garments and other articles).

The term “non-fabric detergent compositions” include non-textile surfacedetergent compositions, including but not limited to compositions forhard surface cleaning, such as dishwashing detergent compositionsincluding manual dish wash compositions, oral detergent compositions,denture detergent compositions, and personal cleansing compositions.

The term “effective amount of enzyme” refers to the quantity of enzymenecessary to achieve the enzymatic activity required in the specificapplication, e.g., in a defined detergent composition. Such effectiveamounts are readily ascertained by one of ordinary skill in the art andare based on many factors, such as the particular enzyme used, thecleaning application, the specific composition of the detergentcomposition, and whether a liquid or dry (e.g., granular, bar)composition is required, and the like. The term “effective amount” of aprotease variant refers to the quantity of protease variant describedhereinbefore that achieves a desired level of enzymatic activity, e.g.,in a defined detergent composition.

The term “water hardness” or “degree of hardness” or “dH” or “° dH” asused herein refers to German degrees of hardness. One degree is definedas 10 milligrams of calcium oxide per liter of water.

The term “relevant washing conditions” is used herein to indicate theconditions, particularly washing temperature, time, washing mechanics,detergent concentration, type of detergent and water hardness, actuallyused in households in a detergent market segment.

The term “adjunct materials” means any liquid, solid or gaseous materialselected for the particular type of detergent composition desired andthe form of the product (e.g., liquid, granule, powder, bar, paste,spray, tablet, gel, or foam composition), which materials are alsopreferably compatible with the protease variant enzyme used in thecomposition. In some embodiments, granular compositions are in “compact”form, while in other embodiments, the liquid compositions are in a“concentrated” form.

The term “stain removing enzyme” as used herein, describes an enzymethat aids the removal of a stain or soil from a fabric or a hardsurface. Stain removing enzymes act on specific substrates, e.g.,protease on protein, amylase on starch, lipase and cutinase on lipids(fats and oils), pectinase on pectin and hemicellulases onhemicellulose. Stains are often depositions of complex mixtures ofdifferent components which either results in a local discoloration ofthe material by itself or which leaves a sticky surface on the objectwhich may attract soils dissolved in the washing liquor therebyresulting in discoloration of the stained area. When an enzyme acts onits specific substrate present in a stain the enzyme degrades orpartially degrades its substrate thereby aiding the removal of soils andstain components associated with the substrate during the washingprocess. For example, when a protease acts on a blood stain it degradesthe protein components in the blood.

The term “reduced amount” means in this context that the amount of thecomponent is smaller than the amount which would be used in a referenceprocess under otherwise the same conditions.

The term “low detergent concentration” system includes detergents whereless than about 800 ppm of detergent components is present in the washwater. Asian, e.g., Japanese detergents are typically considered lowdetergent concentration systems.

The term “medium detergent concentration” system includes detergentswherein between about 800 ppm and about 2000 ppm of detergent componentsis present in the wash water. North American detergents are generallyconsidered to be medium detergent concentration systems.

The term “high detergent concentration” system includes detergentswherein greater than about 2000 ppm of detergent components is presentin the wash water. European detergents are generally considered to behigh detergent concentration systems.

Conventions for Designation of Variants

For purposes of the present invention, the mature polypeptide disclosedin SEQ ID NO: 3 is used to determine the corresponding amino acidresidue in another protease. The amino acid sequence of another proteaseis aligned with the mature polypeptide disclosed in SEQ ID NO: 3, andbased on the alignment, the amino acid position number corresponding toany amino acid residue in the mature polypeptide disclosed in SEQ ID NO:3 is determined using the Needleman-Wunsch algorithm (Needleman andWunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needleprogram of the EMBOSS package (EMBOSS: The European Molecular BiologyOpen Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277),preferably version 5.0.0 or later. The parameters used are gap openpenalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSSversion of BLOSUM62) substitution matrix.

Identification of the corresponding amino acid residue in anotherprotease can be determined by an alignment of multiple polypeptidesequences using several computer programs including, but not limited to,MUSCLE (multiple sequence comparison by log-expectation; version 3.5 orlater; Edgar, 2004, Nucleic Acids Research 32: 1792-1797), MAFFT(version 6.857 or later; Katoh and Kuma, 2002, Nucleic Acids Research30: 3059-3066; Katoh et al., 2005, Nucleic Acids Research 33: 511-518;Katoh and Toh, 2007, Bioinformatics 23: 372-374; Katoh et al., 2009,Methods in Molecular Biology 537:_39-64; Katoh and Toh, 2010,Bioinformatics 26:_1899-1900), and EMBOSS EMMA employing ClustalW (1.83or later; Thompson et al., 1994, Nucleic Acids Research 22: 4673-4680),using their respective default parameters.

When the other enzyme has diverged from the mature polypeptide of SEQ IDNO: 3 such that traditional sequence-based comparison fails to detecttheir relationship (Lindahl and Elofsson, 2000, J. Mol. Biol. 295:613-615), other pairwise sequence comparison algorithms can be used.Greater sensitivity in sequence-based searching can be attained usingsearch programs that utilize probabilistic representations ofpolypeptide families (profiles) to search databases. For example, thePSI-BLAST program generates profiles through an iterative databasesearch process and is capable of detecting remote homologs (Atschul etal., 1997, Nucleic Acids Res. 25: 3389-3402). Even greater sensitivitycan be achieved if the family or superfamily for the polypeptide has oneor more representatives in the protein structure databases. Programssuch as GenTHREADER (Jones, 1999, J. Mol. Biol. 287: 797-815; McGuffinand Jones, 2003, Bioinformatics 19: 874-881) utilize information from avariety of sources (PSI-BLAST, secondary structure prediction,structural alignment profiles, and solvation potentials) as input to aneural network that predicts the structural fold for a query sequence.Similarly, the method of Gough et al., 2000, J. Mol. Biol. 313: 903-919,can be used to align a sequence of unknown structure with thesuperfamily models present in the SCOP database. These alignments can inturn be used to generate homology models for the polypeptide, and suchmodels can be assessed for accuracy using a variety of tools developedfor that purpose.

For proteins of known structure, several tools and resources areavailable for retrieving and generating structural alignments. Forexample the SCOP super families of proteins have been structurallyaligned, and those alignments are accessible and downloadable. Two ormore protein structures can be aligned using a variety of algorithmssuch as the distance alignment matrix (Holm and Sander, 1998, Proteins33: 88-96) or combinatorial extension (Shindyalov and Bourne, 1998,Protein Engineering 11: 739-747), and implementation of these algorithmscan additionally be utilized to query structure databases with astructure of interest in order to discover possible structural homologs(e.g., Holm and Park, 2000, Bioinformatics 16: 566-567).

In describing the variants of the present invention, the nomenclaturedescribed below is adapted for ease of reference. The accepted IUPACsingle letter or three letters amino acid abbreviations are employed.Amino acid positions are indicated with #₁, #₂, etc.

Substitutions:

For an amino acid substitution, the following nomenclature is used:Original amino acid, position, substituted amino acid. Accordingly, thesubstitution of serine at position #₁ with tryptophan is designated as“Ser#₁Trp” or “S#₁W”. Multiple mutations are separated by addition marks(“+”) or by commas (,), e.g., “Ser#₁Trp+“Ser#₂Pro” or S#₁W, S#₂P,representing substitutions at positions #1 and #₂ of serine (S) withtryptophan (W) and proline (P), respectively. If more than one aminoacid may be substituted in a given position these are listed inbrackets, such as [X] or {X}. Thus if both Trp and Lys according to theinvention may be substituted instead of the amino acid occupying atposition #₁ this is indicated as X#₁ {W, K} or X#₂ [W, K] where the Xindicate the amino acid residues of different proteases which accordingto the invention may be parent e.g. such as a protease with SEQ ID NO 3or a protease having at least 70% identity hereto. Thus in some casesthe variants are represented as #₁ {W, K} or X#₂P indicating that theamino acids to be substituted vary depending on the parent. As SEQ ID NO3 is used for numbering the substitutions according to the presentapplication may be indicated with the amino acid present in thecorresponding position in SEQ ID NO 3.

Deletions:

For an amino acid deletion, the following nomenclature is used: Originalamino acid, position, *. Accordingly, the deletion of serine at position#₁ is designated as “Ser#₁*” or “S#₁*” Multiple deletions are separatedby addition marks (“+”) or commas, e.g., “Ser#₁*+Ser#₂*” or “S#₁*,S#₂*”.

Insertions:

The insertion of an additional amino acid residue such as e.g. a lysineafter G#₁ may be indicated by: Gly#₁GlyLys or G#₁GK. Alternativelyinsertion of an additional amino acid residue such as lysine after G#₁may be indicated by: *#₁aL. When more than one amino acid residue isinserted, such as e.g. a Lys, and Ala after #₁ this may be indicated as:Gly#₁GlyLysAla or G#₁GKA. In such cases, the inserted amino acidresidue(s) may also be numbered by the addition of lower case letters tothe position number of the amino acid residue preceding the insertedamino acid residue(s), in this example: *#₁aK *#₁bA.

Multiple Alterations:

Variants comprising multiple alterations are separated by addition marks(“+”) or by commas (,), e.g., “Ser#₁Trp+Ser#₂Pro” or “S#₁W, S#₂P”representing a substitution of serine at positions #₁ and #₂ withtryptophan and proline, respectively as described above.

Different Alterations:

Where different alterations can be introduced at a position, thedifferent alterations are separated by a comma, e.g., “Ser#₁Trp, Lys” orS#₁W, K represents a substitution of serine at position #₁ withtryptophan or lysine. Thus, “Ser#₁Trp, Lys+Ser#₂Asp” designates thefollowing variants: “Ser#₁Trp+Ser#₂Pro”, “Ser#₁Lys+Ser#₂Pro” or S#₁W,K+S#₂D.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides novel protease variants obtained fromBacillus sp., in particular, Bacillus sp. TY-145. The protease variantsof the invention have at least 60% sequence identity to a polypeptidewith SEQ ID NO 3 and comprise a substitution of at least one amino acidposition compared to the protease with SEQ ID NO: 3, the at least oneamino acid position selected from the group consisting of the positions70, 111, 114, 144, 145, 146, 146, 150, 151, 176, 178, 182, 184, 224, and240. One embodiment of the invention relates to protease variants havingat least 60% identity to SEQ ID NO 3, having proteolytic activity andcomprising one or more substitutions selected from the group consistingof Q70F, Q70A, Q70N, S111R, S111E, S111D, S114A, S114Q, S144R, A145E,K146T, K146N, K146W, K146F, K146A, I150A, I150N, I150N, I150S,A151R,N176Y, I178Y, I178F, I178P, G182A, L184F, L184Y, L184F, L184Y, L184W,L184D, R224D, R224G, R224S and Y240R wherein each position correspondsto the position of the polypeptide of SEQ ID NO: 3. In a preferredembodiment the protease variant comprises one or more substitutionsselected from the group consisting of Q70F, Q70A, Q70N, S111R, S111E,S111D, S114A, S114Q, S144R, A145E, K146T, K146N, K146W, K146F, K146A,I150A, I150N, I150N, I150S,A151R, N176Y, I178Y, I178F, I178P, G182A,L184F, L184Y, L184F, L184Y, L184W, L184D, R224D, R224G, R224S, andY240R, wherein each position corresponds to the position of thepolypeptide of SEQ ID NO: 3 and wherein the variant has at least 60%, atleast 70%, at least 75%, at least 76%, at least 77%, at least 78%, atleast 79%, at least 80%, at least 81%, at least 82%, at least 83%, atleast 84%, at least 85%, at least 86%, at least 87%, at least 88%, atleast 89%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95% identity, at least 96%, at least 97%, at least98%, or at least 99%, e. g. at least 99.1%, at least 99.2%, at least99.3%, at least 99.4%, at least 99.5%, at least 99.6, but less than100%, sequence identity to SEQ ID NO: 3.

Enzymes have long been applied in cleaning compositions. The enzymes arebeneficial since each enzyme have specific substrate specificity whichallows removal of various stains. Proteases acts on protein stains andadding proteases helps removing stains comprising proteins such as coco,blood and egg stains. However, a generally encountered problem withproteases in cleaning compositions in particular in liquid cleaningcompositions is the degradation by proteases of other enzymes in thecomposition and of the protease itself. This may lead to reducedstability of the proteases and other enzymes in the composition and anoverall reduced performance of the enzymes in the cleaning compositions.The storage stability of enzymes in cleaning compositions may beimproved by adding protease inhibitors or stabilizers, which arereversible inhibitors of protease activity, e.g., serine proteaseactivity. It is thus advantageous to stabilize proteases by proteaseinhibitors, preferably, by a reversible protease inhibitor. Severalprotease inhibitors have been described in the art and include proteaseinhibitor such as peptide aldehyde, boric acid, or a boronic acid; or aderivative of any of these. Some inhibitors include but are not limitedto boronic acid or a derivative thereof; preferably, phenylboronic acidor a derivative thereof. Phenyl boronic acid derivative of the followingformula:

wherein R is selected from the group consisting of hydrogen, hydroxy,C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkenyl and substitutedC1-C6 alkenyl. Preferably, R is hydrogen, CH3, CH3CH2 or CH3CH2CH2. Theprotease inhibitor (phenyl boronic acid derivative) is4-formyl-phenyl-boronic acid (4-FPBA). The protease inhibitor isselected from the group consisting of:

thiophene-2 boronic acid, thiophene-3 boronic acid, acetamidophenylboronic acid, benzofuran-2 boronic acid, naphtalene-1 boronic acid,naphtalene-2 boronic acid, 2-FPBA, 3-FBPA, 4-FPBA, 1-thianthrene boronicacid, 4-dibenzofuran boronic acid, 5-methylthiophene-2 boronic, acid,thionaphtrene boronic acid, furan-2 boronic acid, furan-3 boronic acid,4,4 biphenyl-diborinic acid, 6-hydroxy-2-naphtalene, 4-(methylthio)phenyl boronic acid, 4 (trimethyl-silyl)phenyl boronic acid,3-bromothiophene boronic acid, 4-methylthiophene boronic acid, 2-naphtylboronic acid, 5-bromothiphene boronic acid, 5-chlorothiophene boronicacid, dimethylthiophene boronic acid, 2-bromophenyl boronic acid,3-chlorophenyl boronic acid, 3-methoxy-2-thiophene, p-methyl-phenylethylboronic acid, 2-thianthrene boronic acid, di-benzothiophene boronicacid, 4-carboxyphenyl boronic acid, 9-anthryl boronic acid, 3,5dichlorophenyl boronic, acid, diphenyl boronic acidanhydride,o-chlorophenyl boronic acid, p-chlorophenyl boronic acid, m-bromophenylboronic acid, p-bromophenyl boronic acid, p-flourophenyl boronic acid,p-tolyl boronic acid, o-tolyl boronic acid, octyl boronic acid, 1,3,5trimethylphenyl boronic acid, 3-chloro-4-flourophenyl boronic acid,3-aminophenyl boronic acid, 3,5-bis-(triflouromethyl) phenyl boronicacid, 2,4 dichlorophenyl boronic acid, 4-methoxyphenyl boronic acid.

Further boronic acid derivatives suitable as protease inhibitors in thecleaning composition are described in U.S. Pat. No. 4,963,655, U.S. Pat.No. 5,159,060, WO 95/12655, WO 95/29223, WO 92/19707, WO 94/04653, WO94/04654, U.S. Pat. No. 5,442,100, U.S. Pat. No. 5,488,157 and U.S. Pat.No. 5,472,628.

The protease inhibitor may also be a peptide aldehyde having the formulaX-B1-B0-H, wherein the groups have the following meaning:

a) H is hydrogen;

b) B0 is a single amino acid residue with L- or D-configuration and withthe formula: NH—CHR′—CO;

c) B1 is a single amino acid residue; and

d) X consists of one or more amino acid residues (preferably one ortwo), optionally comprising an N-terminal protection group. NH—CHR′—CO(B0) is an L or D-amino acid residue, where R′ may be an aliphatic oraromatic side chain, e.g., aralkyl, such as benzyl, where R′ may beoptionally substituted. More particularly, the B0 residue may be bulky,neutral, polar, hydrophobic and/or aromatic. Examples are the D- orL-form of Tyr (p-tyrosine), m-tyrosine, 3,4-dihydroxyphenylalanine, Phe,Val, Met, norvaline (Nva), Leu, lie or norleucine (Nle). In the aboveformula, X-B1-B0-H, the B1 residue may particularly be small, aliphatic,hydrophobic and/or neutral. Examples are alanine (Ala), cysteine (Cys),glycine (Gly), proline (Pro), serine (Ser), threonine (Thr), valine(Val), norvaline (Nva) and norleucine (Nle), particularly alanine,glycine, or valine. X may in particular be one or two amino acidresidues with an optional N-terminal protection group (i.e. the compoundis a tri- or tetrapeptide aldehyde with or without a protection group).Thus, X may be B2, B3-B2, Z-B2, or Z-B3-B2 where B3 and B2 eachrepresents one amino acid residue, and Z is an N-terminal protectiongroup. The B2 residue may in particular be small, aliphatic and/orneutral, e.g., Ala, Gly, Thr, Arg, Leu, Phe or Val. The B3 residue mayin particular be bulky, hydrophobic, neutral and/or aromatic, e.g., Phe,Tyr, Trp, Phenylglycine, Leu, Val, Nva, Nle or lie. The N-terminalprotection group Z (if present) may be selected from formyl, acetyl,benzoyl, trifluoroacetyl, fluoromethoxy carbonyl, methoxysuccinyl,aromatic and aliphatic urethane protecting groups, benzyloxycarbonyl(Cbz), t-butyloxycarbonyl, adamantyloxycarbonyl, p-methoxybenzylcarbonyl (MOZ), benzyl (Bn), p-methoxybenzyl (PMB) or p-methoxyphenyl(PMP), methoxycarbonyl (Moc); methoxyacetyl (Mac); methyl carbamate or amethylamino carbonyl/methyl urea group. In the case of a tripeptidealdehyde with a protection group (i.e. X=Z-B2), Z is preferably a smallaliphatic group, e.g., formyl, acetyl, fluoromethoxy carbonyl,t-butyloxycarbonyl, methoxycarbonyl (Moc); methoxyacetyl (Mac); methylcarbamate or a Methylamino carbonyl/methyl urea group. In the case of atripeptide aldehyde with a protection group (i.e. X=Z-B3-B2), Z ispreferably a bulky aromatic group such as benzoyl, benzyloxycarbonyl,p-methoxybenzyl carbonyl (MOZ), benzyl (Bn), p-methoxybenzyl (PMB) orp-methoxyphenyl (PMP).

Suitable peptide aldehydes are described in WO 94/04651, WO 95/25791, WO98/13458, WO 98/13459, WO 98/13460, WO 98/13461, WO 98/13461, WO98/13462, WO 2007/141736, 2007/145963, WO 2009/118375, WO 2010/055052and WO 2011/036153. More particularly, the peptide aldehyde may beCbz-RAY-H, Ac-GAY-H, Cbz-GAY-H, Cbz-GAL-H, Cbz-VAL-H, Cbz-GAF-H,Cbz-GAV-H, Cbz-GGY-H, Cbz-GGF-H, Cbz-RVY-H, Cbz-LVY-H, Ac-LGAY-H,Ac-FGAY-H, Ac-YGAY-H, Ac-FGAL-H, Ac-FGAF-H, Ac-FGVY-H, Ac-FGAM-H,Ac-WLVY-H, MeO-CO-VAL-H, MeNCO-VAL-H, MeO-CO-FGAL-H, MeO-CO-FGAF-H,MeSO2-FGAL-H, MeSO2-VAL-H, PhCH2O(OH)(O)P-VAL-H, EtSO2-FGAL-H,PhCH2SO2-VAL-H, PhCH2O(OH)(O)P-LAL-H, PhCH2O(OH)(O)P-FAL-H, orMeO(OH)(O)P-LGAL-H. Here, Cbz is benzyloxycarbonyl, Me is methyl, Et isethyl, Ac is acetyl, H is hydrogen, and the other letters representamino acid residues denoted by standard single letter notification(e.g., F=Phe, Y=Tyr, L=Leu).

Alternatively, the peptide aldehyde may have the formula as described inWO 2011/036153:

P—O-(Ai-X′)n-An+1-Q

wherein Q is hydrogen, CH3, CX″3, CHX″2, or CH2X″, wherein X″ is ahalogen atom; wherein one X′ is the “double N-capping group” CO, CO—CO,CS, CS—CS or CS—CO, most preferred urido (CO), and the other X′ arenothing, wherein n=1-10, preferably 2-5, most preferably 2, wherein eachof Ai and An+1 is an amino acid residue having the structure:

—NH—CR″—CO— for a residue to the right of X′ ═—CO—, or

—CO—CR″—NH— for a residue to the left of X′ ═—CO—

wherein R″ is H— or an optionally substituted alkyl or alkylaryl groupwhich may optionally include a hetero atom and may optionally be linkedto the N atom, and wherein P is hydrogen or any C-terminal protectiongroup. Examples of such peptide aldehydes include α-MAPI, β-MAPI,F-urea-RVY-H, F-urea-GGY-H, F-urea-GAF-H, F-urea-GAY-H, F-urea-GAL-H,F-urea-GA-Nva-H, F-urea-GA-Nle-H, Y-urea-RVY-H, Y-urea-GAY-H,F-CS-RVF-H, F-CS-RVY-H, F-CS-GAY-H, Antipain, GE20372A, GE20372B,Chymostatin A, Chymostatin B, and Chymostatin C. Further examples ofpeptide aldehydes are disclosed in WO 2010/055052 and WO 2009/118375, WO94/04651, WO 98/13459, WO 98/13461, WO 98/13462, WO 2007/145963, herebyincorporated by reference.

Alternatively to a peptide aldehyde, the protease inhibitor may be ahydrosulfite adduct having the formula X-B1-NH—CHR—CHOH—SO3M, wherein X,B1 and R are defined as above, and M is H or an alkali metal, preferablyNa or K.

The peptide aldehyde may be converted into a water-soluble hydrosulfiteadduct by reaction with sodium bisulfite, as described in textbooks,e.g., March, J. Advanced Organic Chemistry, fourth edition,Wiley-Interscience, US 1992, p 895.

An aqueous solution of the bisulfite adduct may be prepared by reactingthe corresponding peptide aldehyde with an aqueous solution of sodiumbisulfite (sodium hydrogen sulfite, NaHSO3); potassium bisulfite (KHSO3)by known methods, e.g., as described in WO 98/47523; U.S. Pat. No.6,500,802; U.S. Pat. No. 5,436,229; J. Am. Chem. Soc. (1978) 100, 1228;Org. Synth., Coll. vol. 7: 361.

The molar ratio of the above-mentioned peptide aldehydes (orhydrosulfite adducts) to the protease may be at least 1:1 or 1.5:1 andit may be less than 1000:1, more preferred less than 500:1, even morepreferred from 100:1 to 2:1 or from 20:1 to 2:1, or most preferred, themolar ratio is from 10:1 to 2:1.

Formate salts (e.g., sodium formate) and formic acid have also showngood effects as inhibitor of protease activity. Formate can be usedsynergistically with the above-mentioned protease inhibitors, as shownin WO2013/004635. The formate salts may be present in the detergentcomposition in an amount of at least 0.1% w/w or 0.5% w/w, e.g., atleast 1.0%, at least 1.2% or at least 1.5%. The amount of the salt istypically below 5% w/w, below 4% or below 3%.

WO2007/141736, WO2007/145963 and WO2007/145964 disclose the use of areversible peptide protease inhibitor to stabilize liquid detergentcompositions. US2003/157088 describes compositions containing enzymesstabilized with inhibitors.

The present invention provides protease variants stabilized by proteaseinhibitors such as those mentioned above.

Thus a particular embodiment of the invention relates to variants of aparent protease, wherein protease variant has at least 60% identity toSEQ ID NO 3, having proteolytic activity and comprising one or moresubstitutions selected from the group consisting of Q70F, Q70A, Q70N,S111R, S111E, S111D, S114A, S114Q, S144R, A145E, K146T, K146N, K146W,K146F, K146A, I150A, I150N, I150N, I150S,A151R, N176Y, I178Y, I178F,I178P, G182A, L184F, L184Y, L184F, L184Y, L184W, L184D, R224D, R224G,R224S, and Y240R wherein each position corresponds to the position ofthe polypeptide of SEQ ID NO: 3 and wherein the protease variants haveincreased binding to an inhibitor compared to the parent or compared toSEQ ID NO 3. The binding by an inhibitor is measured as described inexample 2 herein. Another particular embodiment of the relates tovariants of a parent protease, wherein protease variant has at least 60%identity to SEQ ID NO 3, having proteolytic activity and comprising oneor more substitutions selected from the group consisting of Q70F, Q70A,Q70N, S111R, S111E, S111D, S114A, S114Q, S144R, A145E, K146T, K146N,K146W, K146F, K146A, I150A, I150N, I150N, I150S,A151R, N176Y, I178Y,I178F, I178P, G182A, L184F, L184Y, L184F, L184Y, L184W, L184D, R224D,R224G, R224S, and Y240R, wherein each position corresponds to theposition of the SEQ ID NO: 3 and wherein the variants have increasedbinding to an inhibitor compared to parent or compared to SEQ ID NO 3,when measured as described in example 2.

In some further embodiments, the present invention relates to proteasevariants having at least 60% sequence identity to SEQ ID NO 3 and have aKi below 1, such as 0.1; 0.2; 0.3; 0.4; 0.5; 0.6; 0.7; 0.8; 0.9; such asbetween; 0.1 and 0.5 or between 0.2 and 0.7 or between 0.3 and 0.9 orbetween 0.4 and 0.9 when the protease variants are tested as describedin example 2 Another particular embodiment of the invention relates tovariants of a parent protease, wherein protease variant has at least 60%identity to SEQ ID NO 3, having proteolytic activity and comprising oneor more substitutions selected from the group consisting of Q70F, Q70A,Q70N, S111R, S111E, S111D, S114A, S114Q, S144R, A145E, K146T, K146N,K146W, K146F, K146A, I150A, I150N, I150N, I150S,A151R, N176Y, I178Y,I178F, I178P, G182A, L184F, L184Y, L184F, L184Y, L184W, L184D, R224D,R224G, R224S, and Y240R wherein each position corresponds to theposition of the polypeptide of SEQ ID NO: 3 and wherein the proteasevariants have increased binding to an inhibitor compared to theproteases with SEQ ID NO 4 or SEQ ID NO 5, when measured as described inexample 2.

In another aspect, the protease variant according to the inventioncomprises a substitution at one or more (e.g., several) positionscorresponding to positions positions 70, 111, 114, 144, 145, 146, 146,150, 151, 176, 178, 182, 184, 224, and 240 positions 70, 111, 114, 144,145, 146, 146, 150, 151, 176, 178, 182, 184, 224, and 240. In anotheraspect, a variant comprises an alteration at two positions correspondingto any of positions positions 70, 111, 114, 144, 145, 146, 146, 150,151, 176, 178, 182, 184, 224, and 240. In another aspect, a variantcomprises an alteration at three positions corresponding to any ofpositions positions 70, 111, 114, 144, 145, 146, 146, 150, 151, 176,178, 182, 184, 224, and 240. In another aspect, a variant comprises analteration at each position corresponding to positions positions 70,111, 114, 144, 145, 146, 146, 150, 151, 176, 178, 182, 184, 224, and240.

In another aspect, the protease variant comprises or consists of asubstitution at a position corresponding to position 114 of SEQ ID NO 3.In another aspect, the amino acid at a position corresponding toposition 114 is substituted with Gin. In one aspect, the variantcomprises or consists of the substitution S114Q of the polypeptide withSEQ ID NO: 3.

In another aspect, the protease variant comprises or consists of asubstitution at a position corresponding to position 146 of SEQ ID NO 3.In another aspect, the amino acid at a position corresponding toposition 146 is substituted with Asn, Trp, Phe or Ala, such as with Asn,such as with Trp such as with Phe or such as with Ala. In one aspect,the variant comprises or consists of the substitution K146N of thepolypeptide with SEQ ID NO: 3. In one aspect, the variant comprises orconsists of the substitution K146W of the polypeptide with SEQ ID NO: 3.In one aspect, the variant comprises or consists of the substitutionK146F of the polypeptide with SEQ ID NO: 3. In one aspect, the variantcomprises or consists of the substitution K146A of the polypeptide withSEQ ID NO: 3.

In another aspect, the protease variant comprises or consists of asubstitution at a position corresponding to position 150 of SEQ ID NO 3.In another aspect, the amino acid at a position corresponding toposition 150 is substituted with Ala, Asn or Arg, such as with Ala, suchas with Asn or such as with Arg. In one aspect, the variant comprises orconsists of the substitution I150A of the polypeptide with SEQ ID NO: 3.In one aspect, the variant comprises or consists of the substitutionI150N of the polypeptide with SEQ ID NO: 3. In one aspect, the variantcomprises or consists of the substitution I150R of the polypeptide withSEQ ID NO: 3.

In another aspect, the protease variant comprises or consists of asubstitution at a position corresponding to position 151 of SEQ ID NO 3.In another aspect, the amino acid at a position corresponding toposition 151 is substituted with Arg. In one aspect, the variantcomprises or consists of the substitution S151R of the polypeptide withSEQ ID NO: 3.

In another aspect, the protease variant comprises or consists of asubstitution at a position corresponding to position 176 of SEQ ID NO 3.In another aspect, the amino acid at a position corresponding toposition 176 is substituted with Tyr. In one aspect, the variantcomprises or consists of the substitution N176Y of the polypeptide withSEQ ID NO: 3.

In another aspect, the protease variant comprises or consists of asubstitution at a position corresponding to position 178 of SEQ ID NO 3.In another aspect, the amino acid at a position corresponding toposition 178 is substituted with Tyr, Phe or Pro, such as with Tyr, suchas with Phe or such as with Pro. In one aspect, the variant comprises orconsists of the substitution I178Y of the polypeptide with SEQ ID NO: 3.In one aspect, the variant comprises or consists of the substitutionI178F of the polypeptide with SEQ ID NO: 3. In one aspect, the variantcomprises or consists of the substitution I178P of the polypeptide withSEQ ID NO: 3.

In another aspect, the protease variant comprises or consists of asubstitution at a position corresponding to position 184 of SEQ ID NO 3.In another aspect, the amino acid at a position corresponding toposition 184 is substituted with Phe, Tyr, Trp or Asp, such as with Phe,such as with Tyr, such as with Trp or such as with Asp. In one aspect,the variant comprises or consists of the substitution L184F of thepolypeptide with SEQ ID NO: 3. In one aspect, the variant comprises orconsists of the substitution L184Y of the polypeptide with SEQ ID NO: 3.In one aspect, the variant comprises or consists of the substitutionL184W of the polypeptide with SEQ ID NO: 3. In one aspect, the variantcomprises or consists of the substitution L184D of the polypeptide withSEQ ID NO: 3.

In another aspect, the protease variant comprises or consists of asubstitution at a position corresponding to position 224 of SEQ ID NO 3.In another aspect, the amino acid at a position corresponding toposition 224 is substituted with Asp, Gly or Ser, such as with Asp, suchas with Gly or such as with Ser. In one aspect, the variant comprises orconsists of the substitution R224D of the polypeptide with SEQ ID NO: 3.In one aspect, the variant comprises or consists of the substitutionR224G of the polypeptide with SEQ ID NO: 3. In one aspect, the variantcomprises or consists of the substitution R224S of the polypeptide withSEQ ID NO: 3.

In another aspect, the protease variants according to the inventioncomprises or consists of one or more (e.g., several) substitutionsselected from the group consisting of: Q70F, Q70A, Q70N, S111R, S111E,S111D, S114A, S114Q, S144R, A145E, K146T, K146N, K146W, K146F, K146A,I150A, I150N, I150N, I150S,A151R, N176Y, I178Y, I178F, I178P, G182A,L184F, L184Y, L184F, L184Y, L184W, L184D, R224D, R224G, R224S, andY240R.

One aspect of the invention relates to a protease variant of a proteaseparent wherein parent protease is a polypeptide with SEQ ID NO: 3, SEQID NO: 4, SEQ ID NO: 5, or a polypeptide having at least 60%, at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98% or at least 99% identityto the polypeptide with SEQ ID NO: 3 and wherein the protease variantcomprises or consists of the substitution S114Q compared to the parentprotease.

One aspect of the invention relates to a protease variant of a proteaseparent wherein parent protease is a polypeptide with SEQ ID NO: 3, SEQID NO: 4, SEQ ID NO: 5, or a polypeptide having at least 60%, at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98% or at least 99% identityto the polypeptide with SEQ ID NO: 3 and wherein the protease variantcomprises or consists of the substitution Q70F compared to the parentprotease.

One aspect of the invention relates to a protease variant of a proteaseparent wherein parent protease is a polypeptide with SEQ ID NO: 3, SEQID NO: 4, SEQ ID NO: 5, or a polypeptide having at least 60%, at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98% or at least 99% identityto the polypeptide with SEQ ID NO: 3 and wherein the protease variantcomprises or consists of the substitution Q70A compared to the parentprotease.

One aspect of the invention relates to a protease variant of a proteaseparent wherein parent protease is a polypeptide with SEQ ID NO: 3, SEQID NO: 4, SEQ ID NO: 5, or a polypeptide having at least 60%, at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98% or at least 99% identityto the polypeptide with SEQ ID NO: 3 and wherein the protease variantcomprises or consists of the substitution Q70N compared to the parentprotease.

One aspect of the invention relates to a protease variant of a proteaseparent wherein parent protease is a polypeptide with SEQ ID NO: 3, SEQID NO: 4, SEQ ID NO: 5, or a polypeptide having at least 60%, at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98% or at least 99% identityto the polypeptide with SEQ ID NO: 3 and wherein the protease variantcomprises or consists of the substitution S111R compared to the parentprotease.

One aspect of the invention relates to a protease variant of a proteaseparent wherein parent protease is a polypeptide with SEQ ID NO: 3, SEQID NO: 4, SEQ ID NO: 5, or a polypeptide having at least 60%, at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98% or at least 99% identityto the polypeptide with SEQ ID NO: 3 and wherein the protease variantcomprises or consists of the substitution S111E compared to the parentprotease.

One aspect of the invention relates to a protease variant of a proteaseparent wherein parent protease is a polypeptide with SEQ ID NO: 3, SEQID NO: 4, SEQ ID NO: 5, or a polypeptide having at least 60%, at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98% or at least 99% identityto the polypeptide with SEQ ID NO: 3 and wherein the protease variantcomprises or consists of the substitution S111D compared to the parentprotease.

One aspect of the invention relates to a protease variant of a proteaseparent wherein parent protease is a polypeptide with SEQ ID NO: 3, SEQID NO: 4, SEQ ID NO: 5, or a polypeptide having at least 60%, at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98% or at least 99% identityto the polypeptide with SEQ ID NO: 3 and wherein the protease variantcomprises or consists of the substitution S114A compared to the parentprotease.

One aspect of the invention relates to a protease variant of a proteaseparent wherein parent protease is a polypeptide with SEQ ID NO: 3, SEQID NO: 4, SEQ ID NO: 5, or a polypeptide having at least 60%, at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98% or at least 99% identityto the polypeptide with SEQ ID NO: 3 and wherein the protease variantcomprises or consists of the substitution K146T compared to the parentprotease.

One aspect of the invention relates to a protease variant of a proteaseparent wherein parent protease is a polypeptide with SEQ ID NO: 3, SEQID NO: 4, SEQ ID NO: 5, or a polypeptide having at least 60%, at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98% or at least 99% identityto the polypeptide with SEQ ID NO: 3 and wherein the protease variantcomprises or consists of the substitution S144R compared to the parentprotease.

One aspect of the invention relates to a protease variant of a proteaseparent wherein parent protease is a polypeptide with SEQ ID NO: 3, SEQID NO: 4, SEQ ID NO: 5, or a polypeptide having at least 60%, at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98% or at least 99% identityto the polypeptide with SEQ ID NO: 3 and wherein the protease variantcomprises or consists of the substitution A145E compared to the parentprotease.

One aspect of the invention relates to a protease variant of a proteaseparent wherein parent protease is a polypeptide with SEQ ID NO: 3, SEQID NO: 4, SEQ ID NO: 5, or a polypeptide having at least 60%, at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98% or at least 99% identityto the polypeptide with SEQ ID NO: 3 and wherein the protease variantcomprises or consists of the substitution I150N compared to the parentprotease.

One aspect of the invention relates to a protease variant of a proteaseparent wherein parent protease is a polypeptide with SEQ ID NO: 3, SEQID NO: 4, SEQ ID NO: 5, or a polypeptide having at least 60%, at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98% or at least 99% identityto the polypeptide with SEQ ID NO: 3 and wherein the protease variantcomprises or consists of the substitution I150S compared to the parentprotease.

One aspect of the invention relates to a protease variant of a proteaseparent wherein parent protease is a polypeptide with SEQ ID NO: 3, SEQID NO: 4, SEQ ID NO: 5, or a polypeptide having at least 60%, at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98% or at least 99% identityto the polypeptide with SEQ ID NO: 3 and wherein the protease variantcomprises or consists of the substitution G182A compared to the parentprotease.

One aspect of the invention relates to a protease variant of a proteaseparent wherein parent protease is a polypeptide with SEQ ID NO: 3, SEQID NO: 4, SEQ ID NO: 5, or a polypeptide having at least 60%, at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98% or at least 99% identityto the polypeptide with SEQ ID NO: 3 and wherein the protease variantcomprises or consists of the substitution L184F compared to the parentprotease.

One aspect of the invention relates to a protease variant of a proteaseparent wherein parent protease is a polypeptide with SEQ ID NO: 3, SEQID NO: 4, SEQ ID NO: 5, or a polypeptide having at least 60%, at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98% or at least 99% identityto the polypeptide with SEQ ID NO: 3 and wherein the protease variantcomprises or consists of the substitution L184Y compared to the parentprotease.

One aspect of the invention relates to a protease variant of a proteaseparent wherein parent protease is a polypeptide with SEQ ID NO: 3, SEQID NO: 4, SEQ ID NO: 5, or a polypeptide having at least 60%, at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98% or at least 99% identityto the polypeptide with SEQ ID NO: 3 and wherein the protease variantcomprises or consists of the substitution Y240R compared to the parentprotease.

One aspect of the invention relates to a protease variant of a proteaseparent wherein parent protease is a polypeptide with SEQ ID NO: 3, SEQID NO: 4, SEQ ID NO: 5, or a polypeptide having at least 60%, at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98% or at least 99% identityto the polypeptide with SEQ ID NO: 3 and wherein the protease variantcomprises or consists of the substitution K146T compared to the parentprotease.

One aspect of the invention relates to a protease variant of a proteaseparent wherein parent protease is a polypeptide with SEQ ID NO: 3, SEQID NO: 4, SEQ ID NO: 5, or a polypeptide having at least 60%, at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98% or at least 99% identityto the polypeptide with SEQ ID NO: 3 and wherein the protease variantcomprises or consists of the substitution K146N compared to the parentprotease.

One aspect of the invention relates to a protease variant of a proteaseparent wherein parent protease is a polypeptide with SEQ ID NO: 3, SEQID NO: 4, SEQ ID NO: 5, or a polypeptide having at least 60%, at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98% or at least 99% identityto the polypeptide with SEQ ID NO: 3 and wherein the protease variantcomprises or consists of the substitution K146W compared to the parentprotease.

One aspect of the invention relates to a protease variant of a proteaseparent wherein parent protease is a polypeptide with SEQ ID NO: 3, SEQID NO: 4, SEQ ID NO: 5, or a polypeptide having at least 60%, at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98% or at least 99% identityto the polypeptide with SEQ ID NO: 3 and wherein the protease variantcomprises or consists of the substitution K146F compared to the parentprotease.

One aspect of the invention relates to a protease variant of a proteaseparent wherein parent protease is a polypeptide with SEQ ID NO: 3, SEQID NO: 4, SEQ ID NO: 5, or a polypeptide having at least 60%, at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98% or at least 99% identityto the polypeptide with SEQ ID NO: 3 and wherein the protease variantcomprises or consists of the substitution K146A compared to the parentprotease.

One aspect of the invention relates to a protease variant of a proteaseparent wherein parent protease is a polypeptide with SEQ ID NO: 3, SEQID NO: 4, SEQ ID NO: 5, or a polypeptide having at least 60%, at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98% or at least 99% identityto the polypeptide with SEQ ID NO: 3 and wherein the protease variantcomprises or consists of the substitution I150A compared to the parentprotease.

One aspect of the invention relates to a protease variant of a proteaseparent wherein parent protease is a polypeptide with SEQ ID NO: 3, SEQID NO: 4, SEQ ID NO: 5, or a polypeptide having at least 60%, at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98% or at least 99% identityto the polypeptide with SEQ ID NO: 3 and wherein the protease variantcomprises or consists of the substitution I150N compared to the parentprotease.

One aspect of the invention relates to a protease variant of a proteaseparent wherein parent protease is a polypeptide with SEQ ID NO: 3, SEQID NO: 4, SEQ ID NO: 5, or a polypeptide having at least 60%, at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98% or at least 99% identityto the polypeptide with SEQ ID NO: 3 and wherein the protease variantcomprises or consists of the substitution A151R compared to the parentprotease.

One aspect of the invention relates to a protease variant of a proteaseparent wherein parent protease is a polypeptide with SEQ ID NO: 3, SEQID NO: 4, SEQ ID NO: 5, or a polypeptide having at least 60%, at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98% or at least 99% identityto the polypeptide with SEQ ID NO: 3 and wherein the protease variantcomprises or consists of the substitution N176Y compared to the parentprotease.

One aspect of the invention relates to a protease variant of a proteaseparent wherein parent protease is a polypeptide with SEQ ID NO: 3, SEQID NO: 4, SEQ ID NO: 5, or a polypeptide having at least 60%, at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98% or at least 99% identityto the polypeptide with SEQ ID NO: 3 and wherein the protease variantcomprises or consists of the substitution I178Y compared to the parentprotease.

One aspect of the invention relates to a protease variant of a proteaseparent wherein parent protease is a polypeptide with SEQ ID NO: 3, SEQID NO: 4, SEQ ID NO: 5, or a polypeptide having at least 60%, at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98% or at least 99% identityto the polypeptide with SEQ ID NO: 3 and wherein the protease variantcomprises or consists of the substitution I178F compared to the parentprotease.

One aspect of the invention relates to a protease variant of a proteaseparent wherein parent protease is a polypeptide with SEQ ID NO: 3, SEQID NO: 4, SEQ ID NO: 5, or a polypeptide having at least 60%, at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98% or at least 99% identityto the polypeptide with SEQ ID NO: 3 and wherein the protease variantcomprises or consists of the substitution I178P compared to the parentprotease.

One aspect of the invention relates to a protease variant of a proteaseparent wherein parent protease is a polypeptide with SEQ ID NO: 3, SEQID NO: 4, SEQ ID NO: 5, or a polypeptide having at least 60%, at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98% or at least 99% identityto the polypeptide with SEQ ID NO: 3 and wherein the protease variantcomprises or consists of the substitution L184F compared to the parentprotease.

One aspect of the invention relates to a protease variant of a proteaseparent wherein parent protease is a polypeptide with SEQ ID NO: 3, SEQID NO: 4, SEQ ID NO: 5, or a polypeptide having at least 60%, at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98% or at least 99% identityto the polypeptide with SEQ ID NO: 3 and wherein the protease variantcomprises or consists of the substitution F184Y compared to the parentprotease.

One aspect of the invention relates to a protease variant of a proteaseparent wherein parent protease is a polypeptide with SEQ ID NO: 3, SEQID NO: 4 or SEQ ID NO: 5, SEQ ID NO: 4 or SEQ ID NO: 5, or a polypeptidehaving at least 60%, at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98% or at least 99% identity to the polypeptide with SEQ ID NO: 3 andwherein the protease variant comprises or consists of the substitutionL184W compared to the parent protease.

One aspect of the invention relates to a protease variant of a proteaseparent wherein parent protease is a polypeptide with SEQ ID NO: 3, SEQID NO: 4, SEQ ID NO: 5, or a polypeptide having at least 60%, at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98% or at least 99% identityto the polypeptide with SEQ ID NO: 3 and wherein the protease variantcomprises or consists of the substitution L184D compared to the parentprotease.

One aspect of the invention relates to a protease variant of a proteaseparent wherein parent protease is a polypeptide with SEQ ID NO: 3, SEQID NO: 4, SEQ ID NO: 5, or a polypeptide having at least 60%, at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98% or at least 99% identityto the polypeptide with SEQ ID NO: 3 and wherein the protease variantcomprises or consists of the substitution R224D compared to the parentprotease.

One aspect of the invention relates to a protease variant of a proteaseparent wherein parent protease is a polypeptide with SEQ ID NO: 3, SEQID NO: 4, SEQ ID NO: 5, or a polypeptide having at least 60%, at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98% or at least 99% identityto the polypeptide with SEQ ID NO: 3 and wherein the protease variantcomprises or consists of the substitution R224G compared to the parentprotease.

One aspect of the invention relates to a protease variant of a proteaseparent wherein parent protease is a polypeptide with SEQ ID NO: 3, SEQID NO: 4, SEQ ID NO: 5, or a polypeptide having at least 60%, at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98% or at least 99% identityto the polypeptide with SEQ ID NO: 3 and wherein the protease variantcomprises or consists of the substitution R224S compared to the parentprotease.

The present invention also relates to cleaning compositions such asdetergent compositions comprising a protease variant of the invention.In one embodiment the cleaning composition is a liquid or powder laundrydetergent, suitable for e.g. washing at high temperature and/or pH, suchas at or above 40° C. and/or at or above pH 8. In one embodiment thecleaning composition is a liquid or powder laundry detergent, suitablefor e.g. washing at low temperature and/or pH, such as at or below 20°C. and/or pH 6. The detergent may also be formulated as a unit dosedetergent and/or compact detergent optionally with minimum or no water.The detergent may also be a dish wash detergent which is preferablyphosphate-free. The cleaning composition may further comprise at leastone additional enzyme, such as carbohydrate-active enzymes likecarbohydrase, pectinase, mannanase, amylase, cellulase, arabinase,galactanase, xylanase, or proteases such as metalloproteases, lipase, a,cutinase, oxidase, e.g., a laccase, and/or peroxidase.

In general positions within a protease useful for making variants arethose positions wherein at least one substitution leads to a variantexhibiting an improved characteristic as compared to the unchangedprotease The variants may further comprise one or more additionalalterations at one or more (e.g., several) other positions. In aparticular preferred embodiment the protease variants of the invention,further comprises a substitution at one or more positions correspondingto positions 171, 173, 175, 179 or 180 of SEQ ID NO: 3, wherein thevariant has a sequence identity to SEQ ID NO: 3 of at least 60%, and thevariant has protease activity. In an even more preferred embodiment theamino acid at the position corresponding to position 171 of SEQ ID NO: 3is selected from the group consisting of Trp, Lys, Glu, Asn and/or theamino acid at the position corresponding to position 173 of SEQ ID NO: 3is Pro, and/or the amino acid at the position corresponding to position175 of SEQ ID NO: 3 is Ala, Val, Pro, and/or the amino acid at theposition corresponding to position 179 of SEQ ID NO: 3 is selected fromthe group consisting of Cys, Val, Gin, Ser, Thr, Glu, His, Lys, Met,Asn, Tyr and Ala and/or the amino acid at the position corresponding toposition 180 of SEQ ID NO 3 is Tyr. In another preferred embodiment theprotease variants of the invention, further comprises a substitution attwo or more positions corresponding to positions 171, 173, 175, 179 or180 of SEQ ID NO: 3, wherein the variant has a sequence identity to SEQID NO: 3 of at least 60% and less than 100%, and the variant hasprotease activity. at two positions corresponding to any of positions171, 173, 175, 179, and 180. In a preferred embodiment of the inventionthe variants of the invention comprises the mutations S173P+S175P asshown in SEQ ID NO 4. In another preferred embodiment the proteasevariants of the invention comprises the mutations S173P+S175P+F180Y asshown in SEQ ID NO 5.

In a particular embodiment of the invention the protease variants of theinvention comprises the following substitutions compared to SEQ ID NO 3:

S114Q,S173P,S175P,F180Y

K146T,S173P,S175P,F180Y

K146N,S173P,S175P,F180Y

K146W,S173P,S175P,F180Y

K146F,S173P,S175P,F180Y

K146A,S173P,S175P,F180Y

I150A,S173P,S175P,F180Y

I150N,S173P,S175P,F180Y

S27K,I150N,S171N,S173P,G174R,S175P,F180Y,Q198E,T297P

S27K,K146P,S148R,A151R,S171N,S173P,G174R,S175P,F180Y,Q198E,N199K,T297P

S173P,S175P,N176Y,F180Y

S173P,S175P,I178Y,F180Y

S173P,S175P,I178F,F180Y

S173P,S175P,I178F,F180Y

S173P,S175P,I178P,F180Y

S173P,S175P,F180Y,L184F

S173P,S175P,F180Y,L184F

S27K,S171N,S173P,G174R,S175P,F180Y,L184F,Q198E,T297P

S27K,I121V,S171N,S173P,G174R,S175P,F180Y,L184F,Q198E,T297P

S27K,Q70N,G107N,I121V,E127Q,S173P,S175P,F180Y,L184F,Q198E,T297P

S27K,S173P,G174K,S175P,F180Y,L184F,Q198E,N199K,T297P

S27K,S173P,G174K,S175P,F180Y,L184F,Q198E,N199R,T297P

S173P,S175P,F180Y,L184Y

S27K,S171N,S173P,G174R,S175P,F180Y,L184Y,Q198E,T297P

S27K,S173P,G174K,S175P,F180Y,L184Y,Q198E,N199K,T297P

S27K,S173P,G174K,S175P,F180Y,L184Y,Q197K,Q198E,T297P

S173P,S175P,F180Y,L184W

S27K,S171N,S173P,G174R,S175P,F180Y,L184W,Q198E,T297P

S173P,S175P,F180Y,L184D

S173P,S175P,F180Y,R224D

S173P,S175P,F180Y,R224G

S27K,S171N,S173P,G174R,S175P,F180Y,Q198E,N199K,R224G,T297P

S173P,S175P,F180Y,R224S

S27K,I121V,S171N,S173P,G174R,S175P,F180Y,Q198E,R224S,T297P

S27K,S171D,S173P,G174R,S175P,G182A,L184F,Q198E,N199K,R224G,T297P

S27K,S171D,S173P,G174R,S175P,G182A,L184F,Q198E,N199K,T297P

S27K,S171N,S173P,G174R,S175P,F180Y,G182A,L184F,Q198E,N199K,R224G,T297P

S27K,S171N,S173P,G174R,S175P,F80Y,G182A,L184F,Q198E,N199K,T297P

S27K,V162T,S173P,G174K,S175P,F180Y,L184F,Q197K,Q198E,T297P

S27K,V162T,S173P,G174K,S175P,F180Y,Q197K,Q198E,T297P

Q70F

Q70A

Q70N

S111R

S111E

S111D

S114A

S144R

A145E

I150N

I150S

G182A

L184F

L184Y

R224G or

Y240R

The variants may further comprise one or more additional alterations atone or more (e.g., several) other positions. The amino acid changes maybe of a minor nature, that is conservative amino acid substitutions orinsertions that do not significantly affect the folding and/or activityof the protein; small deletions, typically of 1-5 amino acids; smallamino- or carboxyl-terminal extensions, such as an amino-terminalmethionine residue; a small linker peptide of up to 20-25 residues,located at the amino- or carboxyl terminal; or a small extension thatfacilitates purification by changing net charge or another function,such as a poly-histidine tract, an antigenic epitope or a bindingdomain.

Examples of conservative substitutions are within the groups of basicamino acids (arginine, lysine and histidine), acidic amino acids(glutamic acid and aspartic acid), polar amino acids (glutamine andasparagine), hydrophobic amino acids (leucine, isoleucine and valine),aromatic amino acids (phenylalanine, tryptophan and tyrosine), and smallamino acids (glycine, alanine, serine, threonine and methionine). Aminoacid substitutions that do not generally alter specific activity areknown in the art and are described, for example, by H. Neurath and R. L.Hill, 1979, In, The Proteins, Academic Press, New York. Commonsubstitutions are Ala/Ser, Val/Ile, Asp/Glu, Asn/Gln, Thr/Ser, Ala/Gly,Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn,Glu/Gln, Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly.

Alternatively, the amino acid changes are of such a nature that thephysico-chemical properties of the polypeptides are altered. Forexample, amino acid changes may improve the thermal stability of thepolypeptide, alter the substrate specificity, change the pH optimum, andthe like.

Essential amino acids in a polypeptide can be identified according toprocedures known in the art, such as site-directed mutagenesis oralanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244:1081-1085). In the latter technique, single alanine mutations areintroduced at every residue in the molecule, and the resultant variantmolecules are tested for protease activity to identify amino acidresidues that are critical to the activity of the molecule. See also,Hilton et al., 1996, J. Biol. Chem. 271: 4699-4708. The active site ofthe enzyme or other biological interaction can also be determined byphysical analysis of structure, as determined by such techniques asnuclear magnetic resonance, crystallography, electron diffraction, orphotoaffinity labeling, in conjunction with mutation of putative contactsite amino acids. See, for example, de Vos et al., 1992, Science 255:306-312; Smith et al., 1992, J. Mol. Biol. 224: 899-904; Wlodaver etal., 1992, FEBS Lett. 309: 59-64. The identity of essential amino acidscan also be inferred from an alignment with a related polypeptide. Forthe TY-145 protease (SEQ ID NO: 3) the catalytic triad comprising theamino acids D35, H72 and S251 is essential for protease activity of theenzyme.

In an embodiment, the variant has improved catalytic activity comparedto the parent enzyme.

The homology between two amino acid sequences is in this contextdescribed by the parameter “identity” for purposes of the presentinvention, the degree of identity between two amino acid sequences isdetermined using the Needleman-Wunsch algorithm as described above. Theoutput from the routine is besides the amino acid alignment thecalculation of the “Percent Identity” between the two sequences.

Based on this description it is routine for a person skilled in the artto identify suitable homologous proteases, which can be modifiedaccording to the invention.

Substantially homologous parent protease variants may have one or more(several) amino acid substitutions, deletions and/or insertions, in thepresent context the term “one or more” is used interchangeably with theterm “several”. These changes are preferably of a minor nature, that isconservative amino acid substitutions as described above and othersubstitutions that do not significantly affect the three-dimensionalfolding or activity of the protein or polypeptide; small deletions,typically of one to about 30 amino acids; and small amino- orcarboxyl-terminal extensions, such as an amino-terminal methionineresidue, a small linker peptide of up to about 20-25 residues, or asmall extension that facilitates purification (an affinity tag), such asa poly-histidine tract, or protein A (Nilsson et al., 1985, EMBO J. 4:1075; Nilsson et al., 1991, Methods Enzymol. 198: 3. See, also, ingeneral, Ford et al., 1991, Protein Expression and Purification 2:95-107.

Although the changes described above preferably are of a minor nature,such changes may also be of a substantive nature such as fusion oflarger polypeptides of up to 300 amino acids or more both as amino- orcarboxyl-terminal extensions.

The parent protease may comprise or consist of the amino acid sequenceof SEQ ID NO: 3 or an allelic variant thereof; or a fragment thereofhaving protease activity. In one aspect, the parent protease comprisesor consists of the amino acid sequence of SEQ ID NO: 3.

The parent protease may be (a) a polypeptide having at least 60%sequence identity to polypeptide of SEQ ID NO: 3; (b) a polypeptideencoded by a polynucleotide that hybridizes under medium or highstringency conditions with (i) the mature polypeptide coding sequence ofSEQ ID NO: 1, (ii) a sequence encoding the mature polypeptide of SEQ IDNO: 2, or (iii) the full-length complement of (i) or (ii); or (c) apolypeptide encoded by a polynucleotide having at least 60% sequenceidentity to the mature polypeptide coding sequence of SEQ ID NO: 1.

In an aspect, the parent protease has a sequence identity to thepolypeptide with SEQ ID NO: 3 of at least 60%, at least 61%, at least62%, at least 63%, at least 64%, at least 65%, at least 66%, at least67%, at least 68%, at least 69%, at least 70%, at least 71%, at least72%, at least 73%, at least 74%, at least 75%, at least 76%, at least77%, at least 78%, at least 79%, at least 80%, at least 81%, at least82%, at least 83%, at least 84%, at least 85%, at least 86%, at least87%, at least 88%, at least 89%, at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98% or at least 99%.

In one aspect, the amino acid sequence of the parent protease differs byno more than 15 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14 or 15, from the polypeptide with SEQ ID NO: 3.

In another aspect, the parent comprises or consists of the amino acidsequence of SEQ ID NO: 3. In another aspect, the parent comprises orconsists of amino acids 1 to 311 of SEQ ID NO: 2.

In one aspect, the amino acid sequence of the parent protease differs byno more than 15 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14 or 15, from the polypeptide with SEQ ID NO: 4.

In another aspect, the parent comprises or consists of the amino acidsequence of SEQ ID NO: 4.

In one aspect, the amino acid sequence of the parent protease differs byno more than 15 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14 or 15, from the polypeptide with SEQ ID NO: 5.

In another aspect, the parent comprises or consists of the amino acidsequence of SEQ ID NO: 5.

In another aspect, the parent protease is encoded by a polynucleotidethat hybridizes under very low stringency conditions, low stringencyconditions, medium stringency conditions, or high stringency conditions,or very high stringency conditions with (i) the mature polypeptidecoding sequence of SEQ ID NO: 1, (ii) a sequence encoding the maturepolypeptide of SEQ ID NO: 2, or (iii) the full-length complement of (i)or (ii), (Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual,2d edition, Cold Spring Harbor, N.Y.).

The polynucleotide of SEQ ID NO: 1 or a subsequence thereof, as well asthe polypeptide of SEQ ID NO: 3 or a fragment thereof may be used todesign nucleic acid probes to identify and clone DNA encoding a parentfrom strains of different genera or species according to methods wellknown in the art. In particular, such probes can be used forhybridization with the genomic DNA or cDNA of a cell of interest,following standard Southern blotting procedures, in order to identifyand isolate the corresponding gene therein. Such probes can beconsiderably shorter than the entire sequence, but should be at least15, e.g., at least 25, at least 35, or at least 70 nucleotides inlength. Preferably, the nucleic acid probe is at least 100 nucleotidesin length, e.g., at least 200 nucleotides, at least 300 nucleotides, atleast 400 nucleotides, at least 500 nucleotides, at least 600nucleotides, at least 700 nucleotides, at least 800 nucleotides, or atleast 900 nucleotides in length.

Both DNA and RNA probes can be used. The probes are typically labeledfor detecting the corresponding gene (for example, with ³²P, ³H, ³⁵S,biotin, or avidin). Such probes are encompassed by the presentinvention.

A genomic DNA or cDNA library prepared from such other strains may bescreened for DNA that hybridizes with the probes described above andencodes a parent. Genomic or other DNA from such other strains may beseparated by agarose or polyacrylamide gel electrophoresis, or otherseparation techniques. DNA from the libraries or the separated DNA maybe transferred to and immobilized on nitrocellulose or other suitablecarrier material. In order to identify a clone or DNA that hybridizeswith SEQ ID NO: 1 or a subsequence thereof, the carrier material is usedin a Southern blot.

For purposes of the present invention, hybridization indicates that thepolynucleotide hybridizes to a labeled nucleic acid probe correspondingto (i) SEQ ID NO: 1; (ii) the mature polypeptide coding sequence of SEQID NO: 1; (iii) a sequence encoding the mature polypeptide of SEQ ID NO:2; (iv) the full-length complement thereof; or (v) a subsequencethereof; under very low to very high stringency conditions. Molecules towhich the nucleic acid probe hybridizes under these conditions can bedetected using, for example, X-ray film or any other detection meansknown in the art.

In one aspect, the nucleic acid probe is the mature polypeptide codingsequence of SEQ ID NO: 1. In another aspect, the nucleotide acid probeis a 80 to 1140 nucleotides long fragment of SEQ ID NO: 1, e.g. 90, 100,200, 300, 400, 500, 600, 700, 800, 900, 1000 or 1100 nucleotides long.In another aspect, the nucleic acid probe is a polynucleotide thatencodes the polypeptide of SEQ ID NO: 2; the mature polypeptide thereof;or a fragment thereof. In another aspect, the nucleic acid probe is SEQID NO: 1 or a sequence encoding the mature polypeptide of SEQ ID NO: 2.

In another embodiment, the parent is encoded by a polynucleotide havinga sequence identity of at least 60%, such as at least 61%, at least 62%,at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, atleast 68%, at least 69%, at least 70%, at least 71%, at least 72%, atleast 73%, at least 74%, at least 75%, at least 76%, at least 77%, atleast 78%, at least 79%, at least 80%, at least 81%, at least 82%, atleast 83%, at least 84%, at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98% or at least 99% to the mature polypeptide coding sequence ofSEQ ID NO: 1 or a sequence encoding the mature polypeptide of SEQ ID NO:2.

The polypeptide may be a hybrid polypeptide in which a region of onepolypeptide is fused at the N-terminus or the C-terminus of a region ofanother polypeptide.

The parent may be a fusion polypeptide or cleavable fusion polypeptidein which another polypeptide is fused at the N-terminus or theC-terminus of the polypeptide of the present invention. A fusionpolypeptide is produced by fusing a polynucleotide encoding anotherpolypeptide to a polynucleotide of the present invention. Techniques forproducing fusion polypeptides are known in the art, and include ligatingthe coding sequences encoding the polypeptides so that they are in frameand that expression of the fusion polypeptide is under control of thesame promoter(s) and terminator. Fusion polypeptides may also beconstructed using intein technology in which fusion polypeptides arecreated post-translationally (Cooper et al., 1993, EMBO J. 12:2575-2583; Dawson et al., 1994, Science 266: 776-779).

A fusion polypeptide can further comprise a cleavage site between thetwo polypeptides. Upon secretion of the fusion protein, the site iscleaved releasing the two polypeptides. Examples of cleavage sitesinclude, but are not limited to, the sites disclosed in Martin et al.,2003, J. Ind. Microbiol. Biotechnol. 3: 568-576; Svetina et al., 2000,J. Biotechnol. 76: 245-251; Rasmussen-Wilson et al., 1997, Appl.Environ. Microbiol. 63: 3488-3493; Ward et al., 1995, Biotechnology 13:498-503; and Contreras et al., 1991, Biotechnology 9: 378-381; Eaton etal., 1986, Biochemistry 25: 505-512; Collins-Racie et al., 1995,Biotechnology 13: 982-987; Carter et al., 1989, Proteins: Structure,Function, and Genetics 6: 240-248; and Stevens, 2003, Drug DiscoveryWorld 4: 35-48.

The parent may be obtained from organisms of any genus. For purposes ofthe present invention, the term “obtained from” as used herein inconnection with a given source shall mean that the parent encoded by apolynucleotide is produced by the source or by a strain in which thepolynucleotide from the source has been inserted. In one aspect, theparent is secreted extracellularly.

The parent may be a bacterial protease. For example, the parent may be aGram-positive bacterial polypeptide such as a Bacillus, Clostridium,Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus,Staphylococcus, Streptococcus, or Streptomyces protease, or aGram-negative bacterial polypeptide such as a Campylobacter, E. coli,Flavobacterium, Fusobacterium, Helicobacter, Ilyobacter, Neisseria,Pseudomonas, Salmonella, or Ureaplasma protease.

In one aspect, the parent is a Bacillus alkalophilus, Bacillusamyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillusclausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacilluslentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus,Bacillus stearothermophilus, Bacillus subtilis, or Bacillusthuringiensis protease

In one aspect, the parent is a Bacillus sp. protease, e.g., the proteasewith SEQ ID NO: 3 or the mature polypeptide of SEQ ID NO 2.

Strains of these species are readily accessible to the public in anumber of culture collections, such as the American Type CultureCollection (ATCC), Deutsche Sammlung von Mikroorganismen undZellkulturen GmbH (DSMZ), Centraalbureau Voor Schimmelcultures (CBS),and Agricultural Research Service Patent Culture Collection, NorthernRegional Research Center (NRRL).

The parent may be identified and obtained from other sources includingmicroorganisms isolated from nature (e.g., soil, composts, water, etc.)or DNA samples obtained directly from natural materials (e.g., soil,composts, water, etc.) using the above-mentioned probes. Techniques forisolating microorganisms and DNA directly from natural habitats are wellknown in the art. A polynucleotide encoding a parent may then beobtained by similarly screening a genomic DNA or cDNA library of anothermicroorganism or mixed DNA sample. Once a polynucleotide encoding aparent has been detected with the probe(s), the polynucleotide can beisolated or cloned by utilizing techniques that are known to those ofordinary skill in the art (see, e.g., Sambrook et al., 1989, supra).

Preparation of Variants

The present invention also relates to a method for obtaining a proteasevariant having at least one improved property compared to SEQ ID NO 3,comprising

-   -   a) introducing into a parent protease having at least 60%        identity to of SEQ ID NO: 3 one or more of the following        substitutions: Q70F, Q70A, Q70N, S111R, S111E, S111D, S114A,        S114Q, S144R, A145E, K146T, K146N, K146W, K146F, K146A, I150A,        I150N, I150N, I150S, A151R, N176Y, I178Y, I178F, I178P, G182A,        L184F, L184Y, L184F, L184Y, L184W, L184D, R224D, R224G, R224S,        and Y240R, wherein the variant has an amino acid sequence which        is at least 60%, at least 70%, at least 80%, at least 85%, at        least 90% or at least 95% identical to SEQ ID NO: 3; and    -   b) recovering the variant.

The variants can be prepared using any mutagenesis procedure known inthe art, such as site-directed mutagenesis, synthetic gene construction,semi-synthetic gene construction, random mutagenesis, shuffling, etc.

The variants of the invention may also be prepared by procedures such asthose mentioned below.

Site-directed mutagenesis is a technique in which one or more (e.g.,several) mutations are introduced at one or more defined sites in apolynucleotide encoding the parent.

Site-directed mutagenesis can be accomplished in vitro by PCR involvingthe use of oligonucleotide primers containing the desired mutation.Site-directed mutagenesis can also be performed in vitro by cassettemutagenesis involving the cleavage by a restriction enzyme at a site inthe plasmid comprising a polynucleotide encoding the parent andsubsequent ligation of an oligonucleotide containing the mutation in thepolynucleotide. Usually the restriction enzyme that digests the plasmidand the oligonucleotide is the same, permitting sticky ends of theplasmid and the insert to ligate to one another. See, e.g., Scherer andDavis, 1979, Proc. Natl. Acad. Sci. USA 76: 4949-4955; and Barton etal., 1990, Nucleic Acids Res. 18: 7349-4966.

Site-directed mutagenesis can also be accomplished in vivo by methodsknown in the art. See, e.g., U.S. Patent Application Publication No.2004/0171154; Storici et al., 2001, Nature Biotechnol. 19: 773-776; Krenet al., 1998, Nat. Med. 4: 285-290; and Calissano and Macino, 1996,Fungal Genet. Newslett. 43: 15-16.

Synthetic gene construction entails in vitro synthesis of a designedpolynucleotide molecule to encode a polypeptide of interest. Genesynthesis can be performed utilizing a number of techniques, such as themultiplex microchip-based technology described by Tian et al. (2004,Nature 432: 1050-1054) and similar technologies wherein oligonucleotidesare synthesized and assembled upon photo-programmable microfluidicchips.

Single or multiple amino acid substitutions, deletions, and/orinsertions can be made and tested using known methods of mutagenesis,recombination, and/or shuffling, followed by a relevant screeningprocedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988,Science 241: 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA86: 2152-2156; WO 95/17413; or WO 95/22625. Other methods that can beused include error-prone PCR, phage display (e.g., Lowman et al., 1991,Biochemistry 30: 10832-10837; U.S. Pat. No. 5,223,409; WO 92/06204) andregion-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Neret al., 1988, DNA 7: 127).

Mutagenesis/shuffling methods can be combined with high-throughput,automated screening methods to detect activity of cloned, mutagenizedpolypeptides expressed by host cells (Ness et al., 1999, NatureBiotechnology 17: 893-896). Mutagenized DNA molecules that encode activepolypeptides can be recovered from the host cells and rapidly sequencedusing standard methods in the art. These methods allow the rapiddetermination of the importance of individual amino acid residues in apolypeptide.

Semi-synthetic gene construction is accomplished by combining aspects ofsynthetic gene construction, and/or site-directed mutagenesis, and/orrandom mutagenesis, and/or shuffling. Semi-synthetic construction istypified by a process utilizing polynucleotide fragments that aresynthesized, in combination with PCR techniques. Defined regions ofgenes may thus be synthesized de novo, while other regions may beamplified using site-specific mutagenic primers, while yet other regionsmay be subjected to error-prone PCR or non-error prone PCRamplification. Polynucleotide subsequences may then be shuffled.

Polynucleotides

The present invention also relates to isolated polynucleotides encodinga variant of the present invention.

Nucleic Acid Constructs

The present invention also relates to nucleic acid constructs comprisinga polynucleotide encoding a variant of the present invention operablylinked to one or more control sequences that direct the expression ofthe coding sequence in a suitable host cell under conditions compatiblewith the control sequences.

The polynucleotide may be manipulated in a variety of ways to providefor expression of a variant. Manipulation of the polynucleotide prior toits insertion into a vector may be desirable or necessary depending onthe expression vector. The techniques for modifying polynucleotidesutilizing recombinant DNA methods are well known in the art.

The control sequence may be a promoter, a polynucleotide which isrecognized by a host cell for expression of the polynucleotide. Thepromoter contains transcriptional control sequences that mediate theexpression of the variant. The promoter may be any polynucleotide thatshows transcriptional activity in the host cell including variant,truncated, and hybrid promoters, and may be obtained from genes encodingextracellular or intracellular polypeptides either homologous orheterologous to the host cell.

Examples of suitable promoters for directing transcription of thenucleic acid constructs of the present invention in a bacterial hostcell are the promoters obtained from the Bacillus amyloliquefaciensalpha-amylase gene (amyQ), Bacillus licheniformis alpha-amylase gene(amyL), Bacillus licheniformis penicillinase gene (penP), Bacillusstearothermophilus maltogenic amylase gene (amyM), Bacillus subtilislevansucrase gene (sacB), Bacillus subtilis xylA and xylB genes,Bacillus thuringiensis crylIIA gene (Agaisse and Lereclus, 1994,Molecular Microbiology 13: 97-107), E. coli lac operon, E. coli trcpromoter (Egon et al., 1988, Gene 69: 301-315), Streptomyces coelicoloragarase gene (dagA), and prokaryotic beta-lactamase gene (Villa-Kamaroffet al., 1978, Proc. Natl. Acad. Sci. USA 75: 3727-3731), as well as thetac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA 80:21-25). Further promoters are described in “Useful proteins fromrecombinant bacteria” in Gilbert et al., 1980, Scientific American 242:74-94; and in Sambrook et al., 1989, supra. Examples of tandem promotersare disclosed in WO 99/43835.

The control sequence may also be a transcription terminator, which isrecognized by a host cell to terminate transcription. The terminatorsequence is operably linked to the 3′-terminus of the polynucleotideencoding the variant. Any terminator that is functional in the host cellmay be used.

Preferred terminators for bacterial host cells are obtained from thegenes for Bacillus clausii alkaline protease (aprH), Bacilluslicheniformis alpha-amylase (amyL), and Escherichia coli ribosomal RNA(rrnB).

The control sequence may also be an mRNA stabilizer region downstream ofa promoter and upstream of the coding sequence of a gene which increasesexpression of the gene.

Examples of suitable mRNA stabilizer regions are obtained from aBacillus thuringiensis crylllA gene (WO 94/25612) and a Bacillussubtilis SP82 gene (Hue et al., 1995, Journal of Bacteriology 177:3465-3471).

The control sequence may also be a signal peptide coding region thatencodes a signal peptide linked to the N-terminus of a variant anddirects the variant into the cell's secretory pathway. The 5′-end of thecoding sequence of the polynucleotide may inherently contain a signalpeptide coding sequence naturally linked in translation reading framewith the segment of the coding sequence that encodes the variant.Alternatively, the 5′-end of the coding sequence may contain a signalpeptide coding sequence that is foreign to the coding sequence. Aforeign signal peptide coding sequence may be required where the codingsequence does not naturally contain a signal peptide coding sequence.Alternatively, a foreign signal peptide coding sequence may simplyreplace the natural signal peptide coding sequence in order to enhancesecretion of the variant. However, any signal peptide coding sequencethat directs the expressed variant into the secretory pathway of a hostcell may be used.

Effective signal peptide coding sequences for bacterial host cells arethe signal peptide coding sequences obtained from the genes for BacillusNCIB 11837 maltogenic amylase, Bacillus licheniformis subtilisin,Bacillus licheniformis beta-lactamase, Bacillus stearothermophilusalpha-amylase, Bacillus stearothermophilus neutral proteases (nprT,nprS, nprM), and Bacillus subtilis prsA. Further signal peptides aredescribed by Simonen and Palva, 1993, Microbiological Reviews 57:109-137.

The control sequence may also be a propeptide coding sequence thatencodes a propeptide positioned at the N-terminus of a variant. Theresultant polypeptide is known as a proenzyme or propolypeptide (or azymogen in some cases). A propolypeptide is generally inactive and canbe converted to an active polypeptide by catalytic or autocatalyticcleavage of the propeptide from the propolypeptide. The propeptidecoding sequence may be obtained from the genes for Bacillus subtilisalkaline protease (aprE), Bacillus subtilis neutral protease (nprT),Myceliophthora thermophila laccase (WO 95/33836), Rhizomucor mieheiaspartic proteinase, and Saccharomyces cerevisiae alpha-factor.

Where both signal peptide and propeptide sequences are present, thepropeptide sequence is positioned next to the N-terminus of the variantand the signal peptide sequence is positioned next to the N-terminus ofthe propeptide sequence.

It may also be desirable to add regulatory sequences that regulateexpression of the variant relative to the growth of the host cell.Examples of regulatory systems are those that cause expression of thegene to be turned on or off in response to a chemical or physicalstimulus, including the presence of a regulatory compound. Regulatorysystems in prokaryotic systems include the lac, tac, and trp operatorsystems.

Expression Vectors

The present invention also relates to recombinant expression vectorscomprising a polynucleotide encoding a variant of the present invention,a promoter, and transcriptional and translational stop signals. Thevarious nucleotide and control sequences may be joined together toproduce a recombinant expression vector that may include one or moreconvenient restriction sites to allow for insertion or substitution ofthe polynucleotide encoding the variant at such sites. Alternatively,the polynucleotide may be expressed by inserting the polynucleotide or anucleic acid construct comprising the polynucleotide into an appropriatevector for expression. In creating the expression vector, the codingsequence is located in the vector so that the coding sequence isoperably linked with the appropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus) that can be conveniently subjected to recombinant DNA proceduresand can bring about expression of the polynucleotide. The choice of thevector will typically depend on the compatibility of the vector with thehost cell into which the vector is to be introduced. The vector may be alinear or closed circular plasmid.

The vector may be an autonomously replicating vector, i.e., a vectorthat exists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one that, when introduced into the hostcell, is integrated into the genome and replicated together with thechromosome(s) into which it has been integrated. Furthermore, a singlevector or plasmid or two or more vectors or plasmids that togethercontain the total DNA to be introduced into the genome of the host cell,or a transposon, may be used.

The vector preferably contains one or more selectable markers thatpermit easy selection of transformed, transfected, transduced, or thelike cells. A selectable marker is a gene the product of which providesfor biocide or viral resistance, resistance to heavy metals, prototrophyto auxotrophs, and the like.

Examples of bacterial selectable markers are Bacillus licheniformis orBacillus subtilis dal genes, or markers that confer antibioticresistance such as ampicillin, chloramphenicol, kanamycin, neomycin,spectinomycin or tetracycline resistance.

The vector preferably contains an element(s) that permits integration ofthe vector into the host cell's genome or autonomous replication of thevector in the cell independent of the genome.

For integration into the host cell genome, the vector may rely on thepolynucleotide's sequence encoding the variant or any other element ofthe vector for integration into the genome by homologous ornon-homologous recombination. Alternatively, the vector may containadditional polynucleotides for directing integration by homologousrecombination into the genome of the host cell at a precise location(s)in the chromosome(s). To increase the likelihood of integration at aprecise location, the integrational elements should contain a sufficientnumber of nucleic acids, such as 100 to 10,000 base pairs, 400 to 10,000base pairs, and 800 to 10,000 base pairs, which have a high degree ofsequence identity to the corresponding target sequence to enhance theprobability of homologous recombination. The integrational elements maybe any sequence that is homologous with the target sequence in thegenome of the host cell. Furthermore, the integrational elements may benon-encoding or encoding polynucleotides. On the other hand, the vectormay be integrated into the genome of the host cell by non-homologousrecombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. The origin of replication may be any plasmidreplicator mediating autonomous replication that functions in a cell.The term “origin of replication” or “plasmid replicator” means apolynucleotide that enables a plasmid or vector to replicate in vivo.

Examples of bacterial origins of replication are the origins ofreplication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permittingreplication in E. coli, and pUB110, pE194, pTA1060, and pAMß1 permittingreplication in Bacillus.

More than one copy of a polynucleotide of the present invention may beinserted into a host cell to increase production of a variant. Anincrease in the copy number of the polynucleotide can be obtained byintegrating at least one additional copy of the sequence into the hostcell genome or by including an amplifiable selectable marker gene withthe polynucleotide where cells containing amplified copies of theselectable marker gene, and thereby additional copies of thepolynucleotide, can be selected for by cultivating the cells in thepresence of the appropriate selectable agent.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors of the present invention are wellknown to one skilled in the art (see, e.g., Sambrook et al., 1989,supra).

Host Cells

The present invention also relates to recombinant host cells, comprisinga polynucleotide encoding a variant of the present invention operablylinked to one or more control sequences that direct the production of avariant of the present invention. A construct or vector comprising apolynucleotide is introduced into a host cell so that the construct orvector is maintained as a chromosomal integrant or as a self-replicatingextra-chromosomal vector as described earlier. The term “host cell”encompasses any progeny of a parent cell that is not identical to theparent cell due to mutations that occur during replication. The choiceof a host cell will to a large extent depend upon the gene encoding thevariant and its source.

The host cell may be any cell useful in the recombinant production of avariant, e.g., a prokaryote or a eukaryote.

The prokaryotic host cell may be any Gram-positive or Gram-negativebacterium. Gram-positive bacteria include, but are not limited to,Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus,Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, andStreptomyces. Gram-negative bacteria include, but are not limited to,Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter,Ilyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

The bacterial host cell may be any Bacillus cell including, but notlimited to, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillusbrevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans,Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacilluslicheniformis, Bacillus megaterium, Bacillus pumilus, Bacillusstearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells.

The bacterial host cell may also be any Streptococcus cell including,but not limited to, Streptococcus equisimilis, Streptococcus pyogenes,Streptococcus uberis, and Streptococcus equi subsp. Zooepidemicus cells.

The bacterial host cell may also be any Streptomyces cell, including,but not limited to, Streptomyces achromogenes, Streptomyces avermitilis,Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividanscells.

The introduction of DNA into a Bacillus cell may be effected byprotoplast transformation (see, e.g., Chang and Cohen, 1979, Mol. Gen.Genet. 168: 111-115), competent cell transformation (see, e.g., Youngand Spizizen, 1961, J. Bacteriol. 81: 823-829, or Dubnau andDavidoff-Abelson, 1971, J. Mol. Biol. 56: 209-221), electroporation(see, e.g., Shigekawa and Dower, 1988, Biotechniques 6: 742-751), orconjugation (see, e.g., Koehler and Thorne, 1987, J. Bacteriol. 169:5271-5278). The introduction of DNA into an E. coli cell may be effectedby protoplast transformation (see, e.g., Hanahan, 1983, J. Mol. Biol.166: 557-580) or electroporation (see, e.g., Dower et al., 1988, NucleicAcids Res. 16: 6127-6145). The introduction of DNA into a Streptomycescell may be effected by protoplast transformation, electroporation (see,e.g., Gong et al., 2004, Folia Microbiol. (Praha) 49: 399-405),conjugation (see, e.g., Mazodier et al., 1989, J. Bacteriol. 171:3583-3585), or transduction (see, e.g., Burke et al., 2001, Proc. Natl.Acad. Sci. USA 98: 6289-6294). The introduction of DNA into aPseudomonas cell may be effected by electroporation (see, e.g., Choi etal., 2006, J. Microbiol. Methods 64: 391-397), or conjugation (see,e.g., Pinedo and Smets, 2005, Appl. Environ. Microbiol. 71: 51-57). Theintroduction of DNA into a Streptococcus cell may be effected by naturalcompetence (see, e.g., Perry and Kuramitsu, 1981, Infect. Immun. 32:1295-1297), protoplast transformation (see, e.g., Catt and Jollick,1991, Microbios 68: 189-207), electroporation (see, e.g., Buckley etal., 1999, Appl. Environ. Microbiol. 65: 3800-3804) or conjugation (see,e.g., Clewell, 1981, Microbiol. Rev. 45: 409-436). However, any methodknown in the art for introducing DNA into a host cell can be used.

Methods of Production

The present invention also relates to methods of producing a variant,comprising: (a) cultivating a host cell of the present invention underconditions suitable for expression of the variant; and (b) recoveringthe variant.

The host cells are cultivated in a nutrient medium suitable forproduction of the variant using methods known in the art. For example,the cell may be cultivated by shake flask cultivation, or small-scale orlarge-scale fermentation (including continuous, batch, fed-batch, orsolid state fermentations) in laboratory or industrial fermentorsperformed in a suitable medium and under conditions allowing the variantto be expressed and/or isolated. The cultivation takes place in asuitable nutrient medium comprising carbon and nitrogen sources andinorganic salts, using procedures known in the art. Suitable media areavailable from commercial suppliers or may be prepared according topublished compositions (e.g., in catalogues of the American Type CultureCollection). If the variant is secreted into the nutrient medium, thevariant can be recovered directly from the medium. If the variant is notsecreted, it can be recovered from cell lysates.

The variant may be detected using methods known in the art that arespecific for the variants with protease activity. These detectionmethods include, but are not limited to, use of specific antibodies,formation of an enzyme product, or disappearance of an enzyme substrate.For example, an enzyme assay may be used to determine the activity ofthe variant.

The variant may be recovered using methods known in the art. Forexample, the variant may be recovered from the nutrient medium byconventional procedures including, but not limited to, collection,centrifugation, filtration, extraction, spray-drying, evaporation, orprecipitation.

The variant may be purified by a variety of procedures known in the artincluding, but not limited to, chromatography (e.g., ion exchange,affinity, hydrophobic, chromatofocusing, and size exclusion),electrophoretic procedures (e.g., preparative isoelectric focusing),differential solubility (e.g., ammonium sulfate precipitation),SDS-PAGE, or extraction (see, e.g., Protein Purification, Janson andRyden, editors, VCH Publishers, New York, 1989) to obtain substantiallypure variants.

In an alternative aspect, the variant is not recovered, but rather ahost cell of the present invention expressing the variant is used as asource of the variant.

Compositions

Besides enzymes the detergent compositions may comprise additionalcomponents. The choice of additional components is within the skill ofthe artisan and includes conventional ingredients, including theexemplary non-limiting components set forth below. The choice ofcomponents may include, for fabric care, the consideration of the typeof fabric to be cleaned, the type and/or degree of soiling, thetemperature at which cleaning is to take place, and the formulation ofthe detergent product. Although components mentioned below arecategorized by general header according to a particular functionality,this is not to be construed as a limitation, as a component may compriseadditional functionalities as will be appreciated by the skilledartisan.

Detergent Compositions of the Present Invention

The protease variants of the present invention may be added to adetergent composition in an amount corresponding to 0.001-100 mg ofprotein, such as 0.01-100 mg of protein, preferably 0.005-50 mg ofprotein, more preferably 0.01-25 mg of protein, even more preferably0.05-10 mg of protein, most preferably 0.05-5 mg of protein, and evenmost preferably 0.01-1 mg of protein per liter of wash liquor.

The detergent composition may comprise one or more surfactants, whichmay be anionic and/or cationic and/or non-ionic and/or semi-polar and/orzwitterionic, or a mixture thereof. In a particular embodiment, thedetergent composition includes a mixture of one or more nonionicsurfactants and one or more anionic surfactants. The surfactant(s) istypically present at a level of from about 0.1% to 60% by weight, suchas about 1% to about 40%, or about 3% to about 20%, or about 3% to about10%. The surfactant(s) is chosen based on the desired cleaningapplication, and includes any conventional surfactant(s) known in theart. Any surfactant known in the art for use in detergents may beutilized.

When included therein, the detergent will usually contain from about 1%to about 40% by weight, such as from about 5% to about 30%, includingfrom about 5% to about 15%, or from about 20% to about 25% of an anionicsurfactant. Non-limiting examples of anionic surfactants includesulfates and sulfonates, in particular, linear alkylbenzenesulfonates(LAS), isomers of LAS, branched alkylbenzenesulfonates (BABS),phenylalkanesulfonates, alpha-olefinsulfonates (AOS), olefin sulfonates,alkene sulfonates, alkane-2,3-diylbis(sulfates), hydroxyalkanesulfonatesand disulfonates, alkyl sulfates (AS) such as sodium dodecyl sulfate(SDS), fatty alcohol sulfates (FAS), primary alcohol sulfates (PAS),alcohol ethersulfates (AES or AEOS or FES, also known as alcoholethoxysulfates or fatty alcohol ether sulfates), secondaryalkanesulfonates (SAS), paraffin sulfonates (PS), ester sulfonates,sulfonated fatty acid glycerol esters, alpha-sulfo fatty acid methylesters (alpha-SFMe or SES) including methyl ester sulfonate (MES),alkyl- or alkenylsuccinic acid, dodecenyl/tetradecenyl succinic acid(DTSA), fatty acid derivatives of amino acids, diesters and monoestersof sulfo-succinic acid or soap, and combinations thereof.

When included therein, the detergent will usually contain from about 1%to about 40% by weight of a cationic surfactant. Non-limiting examplesof cationic surfactants include alklydimethylehanolamine quat (ADMEAQ),cetyltrimethylammonium bromide (CTAB), dimethyldistearylammoniumchloride (DSDMAC), and alkylbenzyldimethylammonium, and combinationsthereof, Alkyl quaternary ammonium compounds, Alkoxylated quaternaryammonium (AQA),

When included therein, the detergent will usually contain from about0.2% to about 40% by weight of a non-ionic surfactant, for example fromabout 0.5% to about 30%, in particular from about 1% to about 20%, fromabout 3% to about 10%, such as from about 3% to about 5%, or from about8% to about 12%. Non-limiting examples of non-ionic surfactants includealcohol ethoxylates (AE or AEO), alcohol propoxylates, propoxylatedfatty alcohols (PFA), alkoxylated fatty acid alkyl esters, such asethoxylated and/or propoxylated fatty acid alkyl esters, alkylphenolethoxylates (APE), nonylphenol ethoxylates (NPE), alkylpolyglycosides(APG), alkoxylated amines, fatty acid monoethanolamides (FAM), fattyacid diethanolamides (FADA), ethoxylated fatty acid monoethanolamides(EFAM), propoxylated fatty acid monoethanolamide (PFAM), polyhydroxyalkyl fatty acid amides, or N-acyl N-alkyl derivatives of glucosamine(glucamides, GA, or fatty acid glucamide, FAGA), as well as productsavailable under the trade names SPAN and TWEEN, and combinationsthereof.

When included therein, the detergent will usually contain from about 1%to about 40% by weight of a semipolar surfactant. Non-limiting examplesof semipolar surfactants include amine oxides (AO) such asalkyldimethylamineoxide, N-(coco alkyl)-N,N-dimethylamine oxide andN-(tallow-alkyl)-N,N-bis(2-hydroxyethyl)amine oxide, fatty acidalkanolamides and ethoxylated fatty acid alkanolamides, and combinationsthereof.

When included therein, the detergent will usually contain from about 1%to about 40% by weight of a zwitterionic surfactant. Non-limitingexamples of zwitterionic surfactants include betaine,alkyldimethylbetaine, and sulfobetaine, and combinations thereof.

The compositions of the invention may also comprise a hydrotrope whichis a compound that solubilises hydrophobic compounds in aqueoussolutions (or oppositely, polar substances in a non-polar environment).Typically, hydrotropes have both hydrophilic and a hydrophobic character(so-called amphiphilic properties as known from surfactants); howeverthe molecular structure of hydrotropes generally do not favorspontaneous self-aggregation, see e.g. review by Hodgdon and Kaler(2007), Current Opinion in Colloid & Interface Science 12: 121-128.Hydrotropes do not display a critical concentration above whichself-aggregation occurs as found for surfactants and lipids formingmicelles, lamellar or other well defined meso-phases. Instead, manyhydrotropes show a continuous-type aggregation process where the sizesof aggregates grow as concentration increases. However, many hydrotropesalter the phase behavior, stability, and colloidal properties of systemscontaining substances of polar and non-polar character, includingmixtures of water, oil, surfactants, and polymers. Hydrotropes areclassically used across industries from pharma, personal care, food, totechnical applications. Use of hydrotropes in detergent compositionsallow for example more concentrated formulations of surfactants (as inthe process of compacting liquid detergents by removing water) withoutinducing undesired phenomena such as phase separation or high viscosity.

The detergent may contain 0-5% by weight, such as about 0.5 to about 5%,or about 3% to about 5%, of a hydrotrope. Any hydrotrope known in theart for use in detergents may be utilized. Non-limiting examples ofhydrotropes include sodium benzene sulfonate, sodium p-toluenesulfonates (STS), sodium xylene sulfonates (SXS), sodium cumenesulfonates (SCS), sodium cymene sulfonate, amine oxides, alcohols andpolyglycolethers, sodium hydroxynaphthoate, sodium hydroxynaphthalenesulfonate, sodium ethylhexyl sulfate, and combinations thereof.

The detergent composition may contain about 0-65% by weight, such asabout 5% to about 50% of a detergent builder or co-builder, or a mixturethereof. In a dish wash detergent, the level of builder is typically40-65%, particularly 50-65%. The builder and/or co-builder mayparticularly be a chelating agent that forms water-soluble complexeswith Ca and Mg. Any builder and/or co-builder known in the art for usein laundry, ADW and hard surfaces cleaning detergents may be utilized.Non-limiting examples of builders include zeolites, diphosphates(pyrophosphates), triphosphates such as sodium triphosphate (STP orSTPP), carbonates such as sodium carbonate, soluble silicates such assodium metasilicate, layered silicates (e.g., SKS-6 from Hoechst),ethanolamines such as 2-aminoethan-1-ol (MEA), iminodiethanol (DEA) and2,2′,2″-nitrilotriethanol (TEA), and carboxymethylinulin (CMI), andcombinations thereof.

The detergent composition may also contain 0-65% by weight, such asabout 5% to about 40%, of a detergent co-builder, or a mixture thereof.The detergent composition may include a co-builder alone, or incombination with a builder, for example a zeolite builder. Non-limitingexamples of co-builders include homopolymers of polyacrylates orcopolymers thereof, such as poly(acrylic acid) (PAA) or copoly(acrylicacid/maleic acid) (PAA/PMA). Further non-limiting examples includecitrate, chelators such as aminocarboxylates, aminopolycarboxylates andphosphonates, and alkyl- or alkenylsuccinic acid. Additional specificexamples include 2,2′,2″-nitrilotriacetic acid (NTA),etheylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaaceticacid (DTPA), iminodisuccinic acid (IDS), ethylenediamine-N,N′-disuccinicacid (EDDS), methylglycinediacetic acid (MGDA), glutamicacid-N,N-diacetic acid (GLDA), 1-hydroxyethane-1,1-diylbis(phosphonicacid) (HEDP), ethylenediaminetetrakis(methylene)tetrakis(phosphonicacid) (EDTMPA), diethylenetriaminepentakis(methylene)pentakis(phosphonicacid) (DTPMPA), N-(2-hydroxyethyl)iminodiacetic acid (EDG), asparticacid-N-monoacetic acid (ASMA), aspartic acid-N,N-diacetic acid (ASDA),aspartic acid-N-monopropionic acid (ASMP), iminodisuccinic acid (IDA),N-(2-sulfomethyl) aspartic acid (SMAS), N-(2-sulfoethyl) aspartic acid(SEAS), N-(2-sulfomethyl) glutamic acid (SMGL), N-(2-sulfoethyl)glutamic acid (SEGL), N-methyliminodiacetic acid (MIDA),α-alanine-N,N-diacetic acid (α-ALDA), serine-N,N-diacetic acid (SEDA),isoserine-N,N-diacetic acid (ISDA), phenylalanine-N,N-diacetic acid(PHDA), anthranilic acid-N,N-diacetic acid (ANDA), sulfanilic acid-N,N-diacetic acid (SLDA), taurine-N, N-diacetic acid (TUDA) andsulfomethyl-N,N-diacetic acid (SMDA),N-(hydroxyethyl)-ethylidenediaminetriacetate (HEDTA), diethanolglycine(DEG), Diethylenetriamine Penta (Methylene Phosphonic acid) (DTPMP),aminotris(methylenephosphonic acid) (ATMP), and combinations and saltsthereof. Further exemplary builders and/or co-builders are described in,e.g., WO 09/102854, U.S. Pat. No. 5,977,053.

The detergent may contain 0-10% by weight, such as about 1% to about 5%,of a bleaching system. Any bleaching system known in the art for use inlaundry, ADW and hard surfaces cleaning detergents may be utilized.Suitable bleaching system components include bleaching catalysts,photobleaches, bleach activators, sources of hydrogen peroxide such assodium percarbonate and sodium perborates, preformed peracids andmixtures thereof. Suitable preformed peracids include, but are notlimited to, peroxycarboxylic acids and salts, percarbonic acids andsalts, perimidic acids and salts, peroxymonosulfuric acids and salts,for example, Oxone®, and mixtures thereof. Non-limiting examples ofbleaching systems include peroxide-based bleaching systems, which maycomprise, for example, an inorganic salt, including alkali metal saltssuch as sodium salts of perborate (usually mono- or tetra-hydrate),percarbonate, persulfate, perphosphate, persilicate salts, incombination with a peracid-forming bleach activator. By bleach activatoris meant herein a compound which reacts with peroxygen bleach likehydrogen peroxide to form a peracid. The peracid thus formed constitutesthe activated bleach. Suitable bleach activators to be used hereininclude those belonging to the class of esters amides, imides oranhydrides. Suitable examples are tetracetyl athylene diamine (TAED),sodium 3,5,5 trimethyl hexanoyloxybenzene sulphonat, diperoxy dodecanoicacid, 4-(dodecanoyloxy)benzenesulfonate (LOBS),4-(decanoyloxy)benzenesulfonate, 4-(decanoyloxy)benzoate (DOBS),4-(3,5,5-trimethylhexanoyloxy)benzenesulfonate (ISONOBS),tetraacetylethylenediamine (TAED) and 4-(nonanoyloxy)benzenesulfonate(NOBS), and/or those disclosed in WO98/17767. A particular family ofbleach activators of interest was disclosed in EP624154 and particularypreferred in that family is acetyl triethyl citrate (ATC). ATC or ashort chain triglyceride like Triacin has the advantage that it isenvironmental friendly as it eventually degrades into citric acid andalcohol. Furthermore acethyl triethyl citrate and triacetin has a goodhydrolytical stability in the product upon storage and it is anefficient bleach activator. Finally ATC provides a good buildingcapacity to the laundry additive. Alternatively, the bleaching systemmay comprise peroxyacids of, for example, the amide, imide, or sulfonetype. The bleaching system may also comprise peracids such as6-(phthaloylamino)percapronic acid (PAP). The bleaching system may alsoinclude a bleach catalyst. In some embodiments the bleach component maybe an organic catalyst selected from the group consisting of organiccatalysts having the following formulae:

(iii) and mixtures thereof; wherein each R¹ is independently a branchedalkyl group containing from 9 to 24 carbons or linear alkyl groupcontaining from 11 to 24 carbons, preferably each R¹ is independently abranched alkyl group containing from 9 to 18 carbons or linear alkylgroup containing from 11 to 18 carbons, more preferably each R¹ isindependently selected from the group consisting of 2-propylheptyl,2-butyloctyl, 2-pentylnonyl, 2-hexyldecyl, n-dodecyl, n-tetradecyl,n-hexadecyl, n-octadecyl, iso-nonyl, iso-decyl, iso-tridecyl andiso-pentadecyl. Other exemplary bleaching systems are described, e.g.,in WO2007/087258, WO2007/087244, WO2007/087259, WO2007/087242. Suitablephotobleaches may for example be sulfonated zinc phthalocyanine

The cleaning composition may contain 0-10% by weight, such as 0.5-5%,2-5%, 0.5-2% or 0.2-1% of a polymer. Any polymer known in the art foruse in detergents may be utilized. The polymer may function as aco-builder as mentioned above, or may provide antiredeposition, fiberprotection, soil release, dye transfer inhibition, grease cleaningand/or anti-foaming properties. Some polymers may have more than one ofthe above-mentioned properties and/or more than one of thebelow-mentioned motifs. Exemplary polymers include(carboxymethyl)cellulose (CMC), poly(vinyl alcohol) (PVA),poly(vinylpyrrolidone) (PVP), poly(ethyleneglycol) or poly(ethyleneoxide) (PEG), ethoxylated poly(ethyleneimine), carboxymethyl inulin(CMI), and polycarboxylates such as PAA, PAA/PMA, poly-aspartic acid,and lauryl methacrylate/acrylic acid copolymers, hydrophobicallymodified CMC (HM-CMC) and silicones, copolymers of terephthalic acid andoligomeric glycols, copolymers of polyethylene terephthalate andpolyoxyethene terephthalate (PET-POET), PVP, poly(vinylimidazole) (PVI),poly(vinylpyridin-N-oxide) (PVPO or PVPNO) andpolyvinylpyrrolidone-vinylimidazole (PVPVI). Further exemplary polymersinclude sulfonated polycarboxylates, polyethylene oxide andpolypropylene oxide (PEO-PPO) and diquaternium ethoxy sulfate. Otherexemplary polymers are disclosed in, e.g., WO 2006/130575. Salts of theabove-mentioned polymers are also contemplated.

The cleaning compositions may also include fabric hueing agents such asdyes or pigments which when formulated in detergent compositions candeposit onto a fabric when said fabric is contacted with a wash liquorcomprising said detergent compositions thus altering the tint of saidfabric through absorption/reflection of visible light. Fluorescentwhitening agents emit at least some visible light. In contrast, fabrichueing agents alter the tint of a surface as they absorb at least aportion of the visible light spectrum. Suitable fabric hueing agentsinclude dyes and dye-clay conjugates, and may also include pigments.Suitable dyes include small molecule dyes and polymeric dyes. Suitablesmall molecule dyes include small molecule dyes selected from the groupconsisting of dyes falling into the Colour Index (C.I.) classificationsof Direct Blue, Direct Red, Direct Violet, Acid Blue, Acid Red, AcidViolet, Basic Blue, Basic Violet and Basic Red, or mixtures thereof, forexample as described in WO2005/03274, WO2005/03275, WO2005/03276 andEP1876226 (hereby incorporated by reference). A detergent compositionpreferably comprises from about 0.00003 wt % to about 0.2 wt %, fromabout 0.00008 wt % to about 0.05 wt %, or even from about 0.0001 wt % toabout 0.04 wt % fabric hueing agent. The composition may comprise from0.0001 wt % to 0.2 wt % fabric hueing agent, this may be especiallypreferred when the composition is in the form of a unit dose pouch.Suitable hueing agents are also disclosed in, e.g., WO 2007/087257,WO2007/087243.

In one embodiment, the variants according to the invention are combinedwith one or more enzymes, such as at least two enzymes, more preferredat least three, four or five enzymes. Preferably, the enzymes havedifferent substrate specificity, e.g., proteolytic activity, amylolyticactivity, lipolytic activity, hemicellulytic activity or pectolyticactivity.

The detergent additive as well as the detergent composition may compriseone or more additional enzymes such as carbohydrate-active enzymes likecarbohydrase, pectinase, mannanase, amylase, cellulase, arabinase,galactanase, xylanase, or protease, lipase, a, cutinase, oxidase, e.g.,a laccase, and/or peroxidase.

In general, the properties of the selected enzyme(s) should becompatible with the selected detergent, (i.e., pH-optimum, compatibilitywith other enzymatic and non-enzymatic ingredients, etc.), and theenzyme(s) should be present in effective amounts.

Cellulases:

Suitable cellulases include those of bacterial or fungal origin.Chemically modified or protein engineered variants are included.Suitable cellulases include cellulases from the genera Bacillus,Pseudomonas, Humicola, Fusarium, Thielavia, Acremonium, e.g., the fungalcellulases produced from Humicola insolens, Myceliophthora thermophilaand Fusarium oxysporum disclosed in U.S. Pat. No. 4,435,307, U.S. Pat.No. 5,648,263, U.S. Pat. No. 5,691,178, U.S. Pat. No. 5,776,757 and WO89/09259.

Especially suitable cellulases are the alkaline or neutral cellulaseshaving colour care benefits. Examples of such cellulases are cellulasesdescribed in EP 0 495 257, EP 0 531 372, WO 96/11262, WO 96/29397, WO98/08940. Other examples are cellulase variants such as those describedin WO 94/07998, EP 0 531 315, U.S. Pat. No. 5,457,046, U.S. Pat. No.5,686,593, U.S. Pat. No. 5,763,254, WO 95/24471, WO 98/12307 andWO99/001544.

Other cellulases are endo-beta-1,4-glucanase enzyme having a sequence ofat least 97% identity to the amino acid sequence of position 1 toposition 773 of SEQ ID NO:2 of WO 2002/099091 or a family 44xyloglucanase, which a xyloglucanase enzyme having a sequence of atleast 60% identity to positions 40-559 of SEQ ID NO: 2 of WO2001/062903.

Commercially available cellulases include Celluzyme™, and Carezyme™(Novozymes A/S) Carezyme Premium™ (Novozymes A/S), Celluclean™(Novozymes A/S), Celluclean Classic™ (Novozymes A/S), Cellusoft™(Novozymes A/S), Whitezyme™ (Novozymes A/S), Clazinase™, and Puradax HA™(Genencor International Inc.), and KAC-500(B)™ (Kao Corporation).

Mannanases:

Suitable mannanases include those of bacterial or fungal origin.Chemically or genetically modified variants are included. The mannanasemay be an alkaline mannanase of Family 5 or 26. It may be a wild-typefrom Bacillus or Humicola, particularly B. agaradhaerens, B.licheniformis, B. halodurans, B. clausii, or H. insolens. Suitablemannanases are described in WO 1999/064619. A commercially availablemannanase is Mannaway (Novozymes A/S).

Proteases:

Suitable additional proteases include those of bacterial, fungal, plant,viral or animal origin e.g. vegetable or microbial origin. Microbialorigin is preferred. Chemically modified or protein engineered variantsare included. It may be an alkaline protease, such as a serine proteaseor a metalloprotease. A serine protease may for example be of the S1family, such as trypsin, or the S8 family such as subtilisin. Ametalloproteases protease may for example be a thermolysin from e.g.family M4 or other metalloprotease such as those from M5, M7 or M8families.

The term “subtilases” refers to a sub-group of serine protease accordingto Siezen et al., Protein Engng. 4 (1991) 719-737 and Siezen et al.Protein Science 6 (1997) 501-523. Serine proteases are a subgroup ofproteases characterized by having a serine in the active site, whichforms a covalent adduct with the substrate. The subtilases may bedivided into 6 sub-divisions, i.e. the Subtilisin family, the Thermitasefamily, the Proteinase K family, the Lantibiotic peptidase family, theKexin family and the Pyrolysin family.

Examples of subtilases are those derived from Bacillus such as Bacilluslentus, B. alkalophilus, B. subtilis, B. amyloliquefaciens, Bacilluspumilus and Bacillus gibsonii described in; U.S. Pat. No. 7,262,042 andWO09/021867, and subtilisin lentus, subtilisin Novo, subtilisinCarlsberg, Bacillus licheniformis, subtilisin BPN′, subtilisin 309,subtilisin 147 and subtilisin 168 described in WO89/06279 and proteasePD138 described in (WO93/18140). Other useful proteases may be thosedescribed in WO92/175177, WO01/016285, WO02/026024 and WO02/016547.Examples of trypsin-like proteases are trypsin (e.g. of porcine orbovine origin) and the Fusarium protease described in WO89/06270,WO94/25583 and WO05/040372, and the chymotrypsin proteases derived fromCellulomonas described in WO05/052161 and WO05/052146.

A further preferred protease is the alkaline protease from Bacilluslentus DSM 5483, as described for example in WO95/23221, and variantsthereof which are described in WO92/21760, WO95/23221, EP1921147 andEP1921148.

Examples of metalloproteases are the neutral metalloprotease asdescribed in WO07/044993 (Genencor Int.) such as those derived fromBacillus amyloliquefaciens. Examples of useful proteases are thevariants described in: WO92/19729, WO96/034946, WO98/20115, WO98/20116,WO99/011768, WO01/44452, WO03/006602, WO04/03186, WO04/041979,WO07/006305, WO11/036263, WO11/036264, especially the variants withsubstitutions in one or more of the following positions: 3, 4, 9, 15,27, 36, 57, 68, 76, 87, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104,106, 118, 120, 123, 128, 129, 130, 160, 167, 170, 194, 195, 199, 205,206, 217, 218, 222, 224, 232, 235, 236, 245, 248, 252 and 274 using theBPN′ numbering. More preferred the protease variants may comprise themutations: S3T, V4I, S9R, A15T, K27R, *36D, V68A, N76D, N87S,R, *97E,A98S, S99G,D,A, S99AD, S101G,M,R S103A, V104I,Y,N, S106A, G118V,R,H120D,N, N123S, S128L, P129Q, S130A, G160D, Y167A, R170S, A194P, G195E,V199M, V205I, L217D, N218D, M222S, A232V, K235L, Q236H, Q245R, N252K,T274A (using BPN′ numbering).

Suitable commercially available protease enzymes include those soldunder the trade names Alcalase®, Duralase™, Durazym™, Relase®, Relase®Ultra, Savinase®, Savinase® Ultra, Primase®, Polarzyme®, Kannase®,Liquanase®, Liquanase® Ultra, BLAZE®, Ovozyme®, Coronase®, Coronase®Ultra, Neutrase®, Everlase® and Esperase® (Novozymes A/S), those soldunder the tradename Maxatase®, Maxacal®, Maxapem®, Purafect®, PurafectPrime®, Purafect MA®, Purafect Ox®, Purafect OxP®, Puramax®, Properase®,FN2®, FN3®, FN4®, Excellase®, Eraser@, Ultimase®, Opticlean® andOptimase® (Danisco/DuPont), Axapem™ (Gist-Brocases N.V.), BLAP (sequenceshown in FIG. 29 of U.S. Pat. No. 5,352,604) and variants hereof (HenkelAG) and KAP (Bacillus alkalophilus subtilisin) from Kao.

Lipases and Cutinases:

Suitable lipases and cutinases include those of bacterial or fungalorigin. Chemically modified or protein engineered variant enzymes areincluded. Examples include lipase from Thermomyces, e.g. from T.lanuginosus (previously named Humicola lanuginosa) as described inEP258068 and EP305216, cutinase from Humicola, e.g. H. insolens(WO96/13580), lipase from strains of Pseudomonas (some of these nowrenamed to Burkholderia), e.g. P. alcaligenes or P. pseudoalcaligenes(EP218272), P. cepacia (EP331376), P. sp. strain SD705 (WO95/06720 &WO96/27002), P. wisconsinensis (WO96/12012), GDSL-type Streptomyceslipases (WO10/065455), cutinase from Magnaporthe grisea (WO10/107560),cutinase from Pseudomonas mendocina (U.S. Pat. No. 5,389,536), lipasefrom Thermobifida fusca (WO11/084412), Geobacillus stearothermophiluslipase (WO11/084417), lipase from Bacillus subtilis (WO11/084599), andlipase from Streptomyces griseus (WO11/150157) and S. pristinaespiralis(WO12/137147).

Other examples are lipase variants such as those described in EP407225,WO92/05249, WO94/01541, WO94/25578, WO95/14783, WO95/30744, WO95/35381,WO95/22615, WO96/00292, WO97/04079, WO97/07202, WO00/34450, WO00/60063,WO01/92502, WO07/87508 and WO09/109500.

Preferred commercial lipase products include Lipolase™, Lipex™; Lipolex™and Lipoclean™ (Novozymes A/S), Lumafast (originally from Genencor) andLipomax (originally from Gist-Brocades).

Still other examples are lipases sometimes referred to asacyltransferases or perhydrolases, e.g. acyltransferases with homologyto Candida antarctica lipase A (WO10/111143), acyltransferase fromMycobacterium smegmatis (WO05/56782), perhydrolases from the CE 7 family(WO09/67279), and variants of the M. smegmatis perhydrolase inparticular the S54V variant used in the commercial product Gentle PowerBleach from Huntsman Textile Effects Pte Ltd (WO10/100028).

Amylases:

Suitable amylases which can be used together with the protease variantsof the invention may be an alpha-amylase or a glucoamylase and may be ofbacterial or fungal origin. Chemically modified or protein engineeredvariants are included. Amylases include, for example, alpha-amylasesobtained from Bacillus, e.g., a special strain of Bacilluslicheniformis, described in more detail in GB 1,296,839.

Suitable amylases include amylases having SEQ ID NO: 2 in WO 95/10603 orvariants having 90% sequence identity to SEQ ID NO: 3 thereof. Preferredvariants are described in WO 94/02597, WO 94/18314, WO 97/43424 and SEQID NO: 4 of WO 99/019467, such as variants with substitutions in one ormore of the following positions: 15, 23, 105, 106, 124, 128, 133, 154,156, 178, 179, 181, 188, 190, 197, 201, 202, 207, 208, 209, 211, 243,264, 304, 305, 391, 408, and 444.

Different suitable amylases include amylases having SEQ ID NO: 6 in WO02/010355 or variants thereof having 90% sequence identity to SEQ ID NO:6. Preferred variants of SEQ ID NO: 6 are those having a deletion inpositions 181 and 182 and a substitution in position 193.

Other amylases which are suitable are hybrid alpha-amylase comprisingresidues 1-33 of the alpha-amylase derived from B. amyloliquefaciensshown in SEQ ID NO: 6 of WO 2006/066594 and residues 36-483 of the B.licheniformis alpha-amylase shown in SEQ ID NO: 4 of WO 2006/066594 orvariants having 90% sequence identity thereof. Preferred variants ofthis hybrid alpha-amylase are those having a substitution, a deletion oran insertion in one of more of the following positions: G48, T49, G107,H156, A181, N190, M197, I201, A209 and Q264. Most preferred variants ofthe hybrid alpha-amylase comprising residues 1-33 of the alpha-amylasederived from B. amyloliquefaciens shown in SEQ ID NO: 6 of WO2006/066594 and residues 36-483 of SEQ ID NO: 4 are those having thesubstitutions:

M197T;

H156Y+A181T+N190F+A209V+Q264S; or

G48A+T491+G107A+H156Y+A181T+N190F+I201F+A209V+Q264S.

Further amylases which are suitable are amylases having SEQ ID NO: 6 inWO 99/019467 or variants thereof having 90% sequence identity to SEQ IDNO: 6. Preferred variants of SEQ ID NO: 6 are those having asubstitution, a deletion or an insertion in one or more of the followingpositions: R181, G182, H183, G184, N195, I206, E212, E216 and K269.Particularly preferred amylases are those having deletion in positionsR181 and G182, or positions H183 and G184.

Additional amylases which can be used are those having SEQ ID NO: 1, SEQID NO: 3, SEQ ID NO: 2 or SEQ ID NO: 7 of WO 96/023873 or variantsthereof having 90% sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQID NO: 3 or SEQ ID NO: 7. Preferred variants of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO: 3 or SEQ ID NO: 7 are those having a substitution, adeletion or an insertion in one or more of the following positions: 140,181, 182, 183, 184, 195, 206, 212, 243, 260, 269, 304 and 476, using SEQID 2 of WO 96/023873 for numbering. More preferred variants are thosehaving a deletion in two positions selected from 181, 182, 183 and 184,such as 181 and 182, 182 and 183, or positions 183 and 184. Mostpreferred amylase variants of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 7are those having a deletion in positions 183 and 184 and a substitutionin one or more of positions 140, 195, 206, 243, 260, 304 and 476.

Other amylases which can be used are amylases having SEQ ID NO: 2 of WO08/153815, SEQ ID NO: 10 in WO 01/66712 or variants thereof having 90%sequence identity to SEQ ID NO: 2 of WO 08/153815 or 90% sequenceidentity to SEQ ID NO: 10 in WO 01/66712. Preferred variants of SEQ IDNO: 10 in WO 01/66712 are those having a substitution, a deletion or aninsertion in one of more of the following positions: 176, 177, 178, 179,190, 201, 207, 211 and 264.

Further suitable amylases are amylases having SEQ ID NO: 2 of WO09/061380 or variants having 90% sequence identity to SEQ ID NO: 2thereof. Preferred variants of SEQ ID NO: 2 are those having atruncation of the C-terminus and/or a substitution, a deletion or aninsertion in one of more of the following positions: Q87, Q98, S125,N128, T131, T165, K178, R180, S181, T182, G183, M201, F202, N225, S243,N272, N282, Y305, R309, D319, Q320, Q359, K444 and G475. More preferredvariants of SEQ ID NO: 2 are those having the substitution in one ofmore of the following positions: Q87E,R, Q98R, S125A, N128C, T131I,T165I, K178L, T182G, M201L, F202Y, N225E,R, N272E,R, S243Q,A,E,D, Y305R,R309A, Q320R, Q359E, K444E and G475K and/or deletion in position R180and/or S181 or of T182 and/or G183. Most preferred amylase variants ofSEQ ID NO: 2 are those having the substitutions:

N128C+K178L+T182G+Y305R+G475K;

N128C+K178L+T182G+F202Y+Y305R+D319T+G475K;

S125A+N128C+K178L+T182G+Y305R+G475K; or

S125A+N128C+T131I+T165I+K178L+T182G+Y305R+G475K wherein the variants areC-terminally truncated and optionally further comprises a substitutionat position 243 and/or a deletion at position 180 and/or position 181.

Further suitable amylases are amylases having SEQ ID NO: 1 of WO13184577or variants having 90% sequence identity to SEQ ID NO: 1 thereof.Preferred variants of SEQ ID NO: 1 are those having a substitution, adeletion or an insertion in one of more of the following positions:K176, R178, G179, T180, G181, E187, N192, M199, I203, S241, R458, T459,D460, G476 and G477. More preferred variants of SEQ ID NO: 1 are thosehaving the substitution in one of more of the following positions:K176L, E187P, N192FYH, M199L, I203YF, S241QADN, R458N, T459S, D460T,G476K and G477K and/or deletion in position R178 and/or S179 or of T180and/or G181.

Most preferred amylase variants of SEQ ID NO: 1 are those having thesubstitutions:

E187P+I203Y+G476K

E187P+I203Y+R458N+T459S+D460T+G476K,

wherein the variants optionally further comprises a substitution atposition 241 and/or a deletion at position 178 and/or position 179.

Further suitable amylases are amylases having SEQ ID NO: 1 of WO10104675or variants having 90% sequence identity to SEQ ID NO: 1 thereof.Preferred variants of SEQ ID NO: 1 are those having a substitution, adeletion or an insertion in one of more of the following positions: N21,D97, V128 K177, R179, S180, I181, G182, M200, L204, E242, G477 and G478.More preferred variants of SEQ ID NO: 1 are those having thesubstitution in one of more of the following positions: N21D, D97N,V128I K177L, M200L, L204YF, E242QA, G477K and G478K and/or deletion inposition R179 and/or S180 or of I181 and/or G182. Most preferred amylasevariants of SEQ ID NO: 1 are those having the substitutions:

N21D+D97N+V128I

wherein the variants optionally further comprises a substitution atposition 200 and/or a deletion at position 180 and/or position 181.

Other suitable amylases are the alpha-amylase having SEQ ID NO: 12 inWO01/66712 or a variant having at least 90% sequence identity to SEQ IDNO: 12. Preferred amylase variants are those having a substitution, adeletion or an insertion in one of more of the following positions ofSEQ ID NO: 12 in WO01/66712: R28, R118, N174; R181, G182, D183, G184,G186, W189, N195, M202, Y298, N299, K302, S303, N306, R310, N314; R320,H324, E345, Y396, R400, W439, R444, N445, K446, Q449, R458, N471, N484.Particular preferred amylases include variants having a deletion of D183and G184 and having the substitutions R118K, N195F, R320K and R458K, anda variant additionally having substitutions in one or more positionselected from the group: M9, G149, G182, G186, M202, T257, Y295, N299,M323, E345 and A339, most preferred a variant that additionally hassubstitutions in all these positions.

Other examples are amylase variants such as those described inWO2011/098531, WO2013/001078 and WO2013/001087.

Commercially available amylases are Duramyl™, Termamyl™, Fungamyl™,Stainzyme™, Stainzyme Plus™, Natalase™, Liquozyme X and BAN™ (fromNovozymes A/S), and Rapidase™, Purastar™/Effectenz™, Powerase, PreferenzS1000, Preferenz S100 and Preferenz S110 (from Genencor InternationalInc./DuPont).

Peroxidases/Oxidases:

Suitable peroxidases/oxidases include those of plant, bacterial orfungal origin. Chemically modified or protein engineered variants areincluded. Examples of useful peroxidases include peroxidases fromCoprinus, e.g., from C. cinereus, and variants thereof as thosedescribed in WO 93/24618, WO 95/10602, and WO 98/15257.

Commercially available peroxidases include Guardzyme™ (Novozymes A/S).

Other Enzymes:

A protease variant according to the invention may also be combined withadditional enzymes such as pectate lyases e.g. Pectawash™,chlorophyllases etc. The protease variant of the invention may be mixedwith any additional enzyme.

The detergent enzyme(s) may be included in a detergent composition byadding separate additives containing one or more enzymes, or by adding acombined additive comprising all of these enzymes. A detergent additive,i.e., a separate additive or a combined additive, can be formulated, forexample, as a granulate, liquid, slurry, etc. Preferred detergentadditive formulations are granulates, in particular non-dustinggranulates, liquids, in particular stabilized liquids, or slurries.

Non-dusting granulates may be produced, e.g., as disclosed in U.S. Pat.Nos. 4,106,991 and 4,661,452 and may optionally be coated by methodsknown in the art. Examples of waxy coating materials are poly(ethyleneoxide) products (polyethyleneglycol, PEG) with mean molar weights of1000 to 20000; ethoxylated nonylphenols having from 16 to 50 ethyleneoxide units; ethoxylated fatty alcohols in which the alcohol containsfrom 12 to 20 carbon atoms and in which there are 15 to 80 ethyleneoxide units; fatty alcohols; fatty acids; and mono- and di- andtriglycerides of fatty acids. Examples of film-forming coating materialssuitable for application by fluid bed techniques are given in GB1483591. Liquid enzyme preparations may, for instance, be stabilized byadding a polyol such as propylene glycol, a sugar or sugar alcohol,lactic acid or boric acid according to established methods. Protectedenzymes may be prepared according to the method disclosed in EP 238,216.

Adjunct Materials

Any detergent components known in the art for use in laundry detergentsmay also be utilized. Other optional detergent components includeanti-corrosion agents, anti-shrink agents, anti-soil redepositionagents, anti-wrinkling agents, bactericides, binders, corrosioninhibitors, disintegrants/disintegration agents, dyes, enzymestabilizers (including boric acid, borates, CMC, and/or polyols such aspropylene glycol), fabric conditioners including clays,fillers/processing aids, fluorescent whitening agents/opticalbrighteners, foam boosters, foam (suds) regulators, perfumes,soil-suspending agents, softeners, suds suppressors, tarnish inhibitors,and wicking agents, either alone or in combination. Any ingredient knownin the art for use in laundry detergents may be utilized. The choice ofsuch ingredients is well within the skill of the artisan.

Dispersants

The detergent compositions can also contain dispersants. In particularpowdered detergents may comprise dispersants. Suitable water-solubleorganic materials include the homo- or co-polymeric acids or theirsalts, in which the polycarboxylic acid comprises at least two carboxylradicals separated from each other by not more than two carbon atoms.Suitable dispersants are for example described in Powdered Detergents,Surfactant science series volume 71, Marcel Dekker, Inc.

Dye Transfer Inhibiting Agents—

The detergent compositions may also include one or more dye transferinhibiting agents. Suitable polymeric dye transfer inhibiting agentsinclude, but are not limited to, polyvinylpyrrolidone polymers,polyamine N-oxide polymers, copolymers of N-vinylpyrrolidone andN-vinylimidazole, polyvinyloxazolidones and polyvinylimidazoles ormixtures thereof. When present in a subject composition, the dyetransfer inhibiting agents may be present at levels from about 0.0001%to about 10%, from about 0.01% to about 5% or even from about 0.1% toabout 3% by weight of the composition.

Fluorescent Whitening Agent—

A detergent compositions will preferably also contain additionalcomponents that may tint articles being cleaned, such as fluorescentwhitening agent or optical brighteners. Where present the brightener ispreferably at a level of about 0.01% to about 0.5%. Any fluorescentwhitening agent suitable for use in a laundry detergent composition maybe used in the composition. The most commonly used fluorescent whiteningagents are those belonging to the classes of diaminostilbene-sulphonicacid derivatives, diarylpyrazoline derivatives and bisphenyl-distyrylderivatives. Examples of the diaminostilbene-sulphonic acid derivativetype of fluorescent whitening agents include the sodium salts of:4,4′-bis-(2-diethanolamino-4-anilino-s-triazin-6-ylamino)stilbene-2,2′-disulphonate; 4,4′-bis-(2,4-dianilino-s-triazin-6-ylamino)stilbene-2.2′-disulphonate;4,4′-bis-(2-anilino-4(N-methyl-N-2-hydroxy-ethylamino)-s-triazin-6-ylamino)stilbene-2,2′-disulphonate,4,4′-bis-(4-phenyl-2,1,3-triazol-2-yl)stilbene-2,2′-disulphonate;4,4′-bis-(2-anilino-4(1-methyl-2-hydroxy-ethylamino)-s-triazin-6-ylamino)stilbene-2,2′-disulphonate and2-(stilbyl-4″-naptho-1,2′:4,5)-1,2,3-trizole-2″-sulphonate. Preferredfluorescent whitening agents are Tinopal DMS and Tinopal CBS availablefrom Ciba-Geigy AG, Basel, Switzerland. Tinopal DMS is the disodium saltof 4,4′-bis-(2-morpholino-4 anilino-s-triazin-6-ylamino) stilbenedisulphonate. Tinopal CBS is the disodium salt of2,2′-bis-(phenyl-styryl) disulphonate. Also preferred are fluorescentwhitening agents is the commercially available Parawhite KX, supplied byParamount Minerals and Chemicals, Mumbai, India. Other fluorescerssuitable for use include the 1-3-diaryl pyrazolines and the7-alkylaminocoumarins.

Suitable fluorescent brightener levels include lower levels of fromabout 0.01, from 0.05, from about 0.1 or even from about 0.2 wt % toupper levels of 0.5 or even 0.75 wt %.

Soil Release Polymers—

The detergent composition may also include one or more soil releasepolymers which aid the removal of soils from fabrics such as cotton andpolyester based fabrics, in particular the removal of hydrophobic soilsfrom polyester based fabrics. The soil release polymers may for examplebe nonionic or anionic terephthalte based polymers, polyvinylcaprolactam and related copolymers, vinyl graft copolymers, polyesterpolyamides see for example Chapter 7 in Powdered Detergents, Surfactantscience series volume 71, Marcel Dekker, Inc. Another type of soilrelease polymers are amphiphilic alkoxylated grease cleaning polymerscomprising a core structure and a plurality of alkoxylate groupsattached to that core structure. The core structure may comprise apolyalkylenimine structure or a polyalkanolamine structure as describedin detail in WO 2009/087523 (hereby incorporated by reference).Furthermore random graft co-polymers are suitable soil release polymersSuitable graft co-polymers are described in more detail in WO2007/138054, WO 2006/108856 and WO 2006/113314 (hereby incorporated byreference). Other soil release polymers are substituted polysaccharidestructures especially substituted cellulosic structures such as modifiedcellulose deriviatives such as those described in EP 1867808 or WO2003/040279 (both are hereby incorporated by reference). Suitablecellulosic polymers include cellulose, cellulose ethers, celluloseesters, cellulose amides and mixtures thereof. Suitable cellulosicpolymers include anionically modified cellulose, nonionically modifiedcellulose, cationically modified cellulose, zwitterionically modifiedcellulose, and mixtures thereof. Suitable cellulosic polymers includemethyl cellulose, carboxy methyl cellulose, ethyl cellulose, hydroxylethyl cellulose, hydroxyl propyl methyl cellulose, ester carboxy methylcellulose, and mixtures thereof.

Anti-Redeposition Agents—

The detergent compositions may also include one or moreanti-redeposition agents such as carboxymethylcellulose (CMC), polyvinylalcohol (PVA), polyvinylpyrrolidone (PVP), polyoxyethylene and/orpolyethyleneglycol (PEG), homopolymers of acrylic acid, copolymers ofacrylic acid and maleic acid, and ethoxylated polyethyleneimines. Thecellulose based polymers described under soil release polymers above mayalso function as anti-redeposition agents.

Other suitable adjunct materials include, but are not limited to,anti-shrink agents, anti-wrinkling agents, bactericides, binders,carriers, dyes, enzyme stabilizers, fabric softeners, fillers, foamregulators, hydrotropes, perfumes, pigments, sod suppressors, solvents,structurants for liquid detergents and/or structure elasticizing agents.

Formulation of Detergent Products

The detergent composition may be in any convenient form, e.g., a bar, ahomogenous tablet, a tablet having two or more layers, a regular orcompact powder, a granule, a paste, a gel, or a regular, compact orconcentrated liquid.

Detergent formulation forms: Layers (same or different phases), Pouches,versus forms for Machine dosing unit.

Pouches can be configured as single or multicompartments. It can be ofany form, shape and material which is suitable for hold the composition,e.g. without allowing the release of the composition to release of thecomposition from the pouch prior to water contact. The pouch is madefrom water soluble film which encloses an inner volume. Said innervolume can be divided into compartments of the pouch. Preferred filmsare polymeric materials preferably polymers which are formed into a filmor sheet. Preferred polymers, copolymers or derivates thereof areselected polyacrylates, and water soluble acrylate copolymers, methylcellulose, carboxy methyl cellulose, sodium dextrin, ethyl cellulose,hydroxyethyl cellulose, hydroxypropyl methyl cellulose, malto dextrin,poly methacrylates, most preferably polyvinyl alcohol copolymers and,hydroxyprpyl methyl cellulose (HPMC). Preferably the level of polymer inthe film for example PVA is at least about 60%. Preferred averagemolecular weight will typically be about 20,000 to about 150,000. Filmscan also be of blend compositions comprising hydrolytically degradableand water soluble polymer blends such as polyactide and polyvinylalcohol (known under the Trade reference M8630 as sold by Chris CraftIn. Prod. Of Gary, Ind., US) plus plasticisers like glycerol, ethyleneglycerol, Propylene glycol, sorbitol and mixtures thereof. The pouchescan comprise a solid laundry cleaning composition or part componentsand/or a liquid cleaning composition or part components separated by thewater soluble film. The compartment for liquid components can bedifferent in composition than compartments containing solids. Ref:(US2009/0011970 A1)

Detergent ingredients can be separated physically from each other bycompartments in water dissolvable pouches or in different layers oftablets. Thereby negative storage interaction between components can beavoided. Different dissolution profiles of each of the compartments canalso give rise to delayed dissolution of selected components in the washsolution.

Definition/Characteristics of the Forms

A liquid or gel detergent, which is not unit dosed, may be aqueous,typically containing at least 20% by weight and up to 95% water, such asup to about 70% water, up to about 65% water, up to about 55% water, upto about 45% water, up to about 35% water. Other types of liquids,including without limitation, alkanols, amines, diols, ethers andpolyols may be included in an aqueous liquid or gel. An aqueous liquidor gel detergent may contain from 0-30% organic solvent.

A liquid or gel detergent may be non-aqueous.

Methods and Uses

The protease variants of the present invention may be added to and thusbecome a component of a detergent composition, wherein said variantcomprises one or more of the following substitutions: Q70F, Q70A, Q70N,S111R, S111E, S111D, S114A, S114Q, S144R, A145E, K146T, K146N, K146W,K146F, K146A, I150A, I150N, I150N, I150S,A151R, N176Y, I178Y, I178F,I178P, G182A, L184F, L184Y, L184F, L184Y, L184W, L184D, R224D, R224G,R224S, and Y240R, of SEQ ID NO: 3, wherein the variant has at least 60%,such as at least 61%, at least 62%, at least 63%, at least 64%, at least65%, at least 66%, at least 67%, at least 68%, at least 69%, at least70%, at least 71%, at least 72%, at least 73%, at least 74%, at least75%, at least 76%, at least 77%, at least 78%, at least 79%, at least80%, at least 81%, at least 82%, at least 83%, at least 84%, at least85%, at least 86%, at least 87%, at least 88%, at least 89%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98% or at least 99% sequenceidentity to SEQ ID NO: 3.

Detergent compositions are generally used in cleaning processes such aslaundry and/or hard surface cleaning e.g. dish wash.

One embodiment of the invention relates to a detergent composition, suchas a laundry or dish wash compositions comprising a protease variant ofa protease parent having at least 60% identity to SEQ ID NO 3 whereinsaid variant comprises at least one substitution selected from the groupconsisting of Q70F, Q70A, Q70N, S111R, S111E, S111D, S114A, S114Q,S144R, A145E, K146T, K146N, K146W, K146F, K146A, I150A, I150N, I150N,I150S,A151R, N176Y, I178Y, I178F, I178P, G182A, L184F, L184Y, L184F,L184Y, L184W, L184D, R224D, R224G, R224S, and Y240R.

A detergent composition may comprise at least one protease variantwherein said variant comprises one or more of the followingsubstitutions Q70F, Q70A, Q70N, S111R, S111E, S111D, S114A, S114Q,S144R, A145E, K146T, K146N, K146W, K146F, K146A, I150A, I150N, I150N,I150S,A151R, N176Y, I178Y, I178F, I178P, G182A, L184F, L184Y, L184F,L184Y, L184W, L184D, R224D, R224G, R224S, and Y240R of SEQ ID NO: 3,wherein the protease variant has a sequence identity to SEQ ID NO: 3 ofat least 60% such as at least at least 61%, at least 62%, at least 63%,at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, atleast 69%, at least 70%, at least 71%, at least 72%, at least 73%, atleast 74%, at least 75%, at least 76%, at least 77%, at least 78%, atleast 79%, at least 80%, at least 81%, at least 82%, at least 83%, atleast 84%, at least 85%, at least 86%, at least 87%, at least 88%, atleast 89%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98% or atleast 99% sequence identity to SEQ ID NO: 3 and the variant has proteaseactivity. The at least one protease variant preferably has increasedinhibition by inhibitor as described under “Material and Methods”.

A detergent composition may be formulated, for example, as a hand ormachine laundry detergent composition including a laundry additivecomposition suitable for pre-treatment of stained fabrics and a rinseadded fabric softener composition, or be formulated as a detergentcomposition for use in general household hard surface cleaningoperations, or be formulated for hand or machine dishwashing operations.

A cleaning process or the textile care process may for example be alaundry process, a dishwashing process or cleaning of hard surfaces suchas bathroom tiles, floors, table tops, drains, sinks and washbasins.Laundry processes can for example be household laundering, but it mayalso be industrial laundering. A process for laundering of fabricsand/or garments may be a process comprises treating fabrics with awashing solution containing a detergent composition, and at least oneprotease variant. A cleaning process or a textile care process can forexample be carried out in a machine washing process or in a manualwashing process. The washing solution can for example be an aqueouswashing solution containing a detergent composition.

The fabrics and/or garments subjected to a washing, cleaning or textilecare process may be conventional washable laundry, for example householdlaundry. Preferably, the major part of the laundry is garments andfabrics, including knits, woven, denims, non-woven, felts, yarns, andtowelling. The fabrics may be cellulose based such as naturalcellulosics, including cotton, flax, linen, jute, ramie, sisal or coiror manmade cellulosics (e.g., originating from wood pulp) includingviscose/rayon, ramie, cellulose acetate fibers (tricell), lyocell orblends thereof. The fabrics may also be non-cellulose based such asnatural polyamides including wool, camel, cashmere, mohair, rabbit andsilk or synthetic polymer such as nylon, aramid, polyester, acrylic,polypropylen and spandex/elastane, or blends thereof as well as blend ofcellulose based and non-cellulose based fibers. Examples of blends areblends of cotton and/or rayon/viscose with one or more companionmaterial such as wool, synthetic fibers (e.g., polyamide fibers, acrylicfibers, polyester fibers, polyvinyl alcohol fibers, polyvinyl chloridefibers, polyurethane fibers, polyurea fibers, aramid fibers), andcellulose-containing fibers (e.g., rayon/viscose, ramie, flax, linen,jute, cellulose acetate fibers, lyocell).

The last few years there has been an increasing interest in replacingcomponents in detergents, which is derived from petrochemicals withrenewable biological components such as enzymes and polypeptides withoutcompromising the wash performance. When the components of detergentcompositions change new enzyme activities or new enzymes havingalternative and/or improved properties compared to the common useddetergent enzymes such as proteases, lipases and amylases is needed toachieve a similar or improved wash performance when compared to thetraditional detergent compositions.

Proteases and variants hereof are usable in proteinaceous stain removingprocesses. The proteinaceous stains may be stains such as food stains,e.g., baby food, sebum, cocoa, egg, blood, milk, ink, grass, or acombination hereof.

Typical detergent compositions include various components in addition tothe enzymes, these components have different effects, some componentslike the surfactants lower the surface tension in the detergent, whichallows the stain being cleaned to be lifted and dispersed and thenwashed away, other components like bleach systems remove discolor oftenby oxidation and many bleaches also have strong bactericidal properties,and are used for disinfecting and sterilizing. Yet other components likebuilder and chelator softens, e.g., the wash water by removing the metalions form the liquid.

The enzyme compositions may further comprise at least one or more of thefollowing: a surfactant, a builder, a chelator or chelating agent,bleach system or bleach component in laundry or dish wash.

The amount of a surfactant, a builder, a chelator or chelating agent,bleach system and/or bleach component may be reduced compared to amountof surfactant, builder, chelator or chelating agent, bleach systemand/or bleach component used without the added protease variant of theinvention. Preferably the at least one component which is a surfactant,a builder, a chelator or chelating agent, bleach system and/or bleachcomponent is present in an amount that is 1% less, such as 2% less, suchas 3% less, such as 4% less, such as 5% less, such as 6% less, such as7% less, such as 8% less, such as 9% less, such as 10% less, such as 15%less, such as 20% less, such as 25% less, such as 30% less, such as 35%less, such as 40% less, such as 45% less, such as 50% less than theamount of the component in the system without the addition of proteasevariants of the invention, such as a conventional amount of suchcomponent. Detergent compositions may also be composition which is freeof at least one component which is a surfactant, a builder, a chelatoror chelating agent, bleach system or bleach component and/or polymer.

Washing Methods

Detergent compositions are ideally suited for use in laundryapplications. These methods include a method for laundering a fabric.The method comprises the steps of contacting a fabric to be launderedwith a cleaning laundry solution comprising a detergent composition. Thefabric may comprise any fabric capable of being laundered in normalconsumer use conditions. The solution preferably has a pH from about 5.5to about 11.5. The compositions may be employed at concentrations fromabout 100 ppm, preferably 500 ppm to about 15,000 ppm in solution. Thewater temperatures typically range from about 5° C. to about 95° C.,including about 10° C., about 15° C., about 20° C., about 25° C., about30° C., about 35° C., about 40° C., about 45° C., about 50° C., about55° C., about 60° C., about 65° C., about 70° C., about 75° C., about80° C., about 85° C. and about 90° C. The water to fabric ratio istypically from about 1:1 to about 30:1.

In particular embodiments, the washing method is conducted at a pH fromabout 5.0 to about 11.5, or from about 6 to about 10.5, about 5 to about11, about 5 to about 10, about 5 to about 9, about 5 to about 8, about 5to about 7, about 5.5 to about 11, about 5.5 to about 10, about 5.5 toabout 9, about 5.5 to about 8, about 5.5. to about 7, about 6 to about11, about 6 to about 10, about 6 to about 9, about 6 to about 8, about 6to about 7, about 6.5 to about 11, about 6.5 to about 10, about 6.5 toabout 9, about 6.5 to about 8, about 6.5 to about 7, about 7 to about11, about 7 to about 10, about 7 to about 9, or about 7 to about 8,about 8 to about 11, about 8 to about 10, about 8 to about 9, about 9 toabout 11, about 9 to about 10, about 10 to about 11, preferably about5.5 to about 11.5.

In particular embodiments, the washing method is conducted at a degreeof hardness of from about 0° dH to about 30° dH, such as about 1° dH,about 2° dH, about 3° dH, about 4° dH, about 5° dH, about 6° dH, about7° dH, about 8° dH, about 9° dH, about 10° dH, about 11° dH, about 12°dH, about 13° dH, about 14° dH, about 15° dH, about 16° dH, about 17°dH, about 18° dH, about 19° dH, about 20° dH, about 21° dH, about 22°dH, about 23° dH, about 24° dH, about 25° dH, about 26° dH, about 27°dH, about 28° dH, about 29° dH, about 30° dH. Under typical Europeanwash conditions, the degree of hardness is about 16° dH, under typicalUS wash conditions about 6° dH, and under typical Asian wash conditions,about 3° dH.

The compositions for use in the methods described above may furthercomprises at least one additional enzyme as set forth in the “otherenzymes” section above, such as an enzyme selected from the group ofhydrolases such as proteases, lipases and cutinases, carbohydrases suchas amylases, cellulases, hemicellulases, xylanases, and pectinase or acombination hereof.

The present invention is further described by the following examplesthat should not be construed as limiting the scope of the invention.

EXAMPLES Materials and Methods Protease Activity Assay 1) Suc-AAPF-pNAActivity Assay:

The proteolytic activity can be determined by a method employing theSuc-AAPF-pNA substrate. Suc-AAPF-pNA is an abbreviation forN-Succinyl-Alanine-Alanine-Proline-Phenylalanine-p-Nitroanilide, and itis a blocked peptide which can be cleaved by endo-proteases. Followingcleavage a free pNA molecule is liberated and it has a yellow colour andthus can be measured by visible spectrophotometry at wavelength 405 nm.The Suc-AAPF-PNA substrate is manufactured by Bachem (cat. no. L1400,dissolved in DMSO).

The protease sample to be analyzed was diluted in residual activitybuffer (100 mM Tris pH 8.6). The assay was performed by transferring 30μl of diluted enzyme samples to 96 well microtiter plate and adding 70μl substrate working solution (0.72 mg/ml in 100 mM Tris pH8.6). Thesolution was mixed at room temperature and absorption is measured every20 sec. over 5 minutes at OD 405 nm. The slope (absorbance per minute)of the time dependent absorption-curve is directly proportional to theactivity of the protease in question under the given set of conditions.The protease sample should be diluted to a level where the slope islinear.

Example 1: Construction of TY145 Variants by Site-Directed Mutagenesis

Site-directed variants were constructed of the TY145 protease (SEQ IDNO: 3) comprising specific substitutions according to the invention. Thevariants were made by traditional cloning of DNA fragments (Sambrook etal., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold SpringHarbor, 1989) using PCR together with properly designed mutagenicoligonucleotides that introduced the desired mutations in the resultingsequence.

Mutagenic oligos were designed corresponding to the DNA sequenceflanking the desired site(s) of mutation, separated by the DNA basepairs defining the insertions/deletions/substitutions, and purchasedfrom an oligo vendor such as Life Technologies. In this manner, thevariants listed in Table 1 below were constructed and produced.

In order to test TY145 protease variants of the invention, the mutatedDNA comprising a variant of the invention were transformed into acompetent B. subtilis strain and fermented using standard protocols(liquid media, 3-4 days, 30° C.). The culture broth was centrifuged(26000×g, 20 min) and the supernatant was carefully decanted from theprecipitate. The supernatant was filtered through a Nalgene 0.2 μmfiltration unit in order to remove the rest of the Bacillus host cells.The 0.2 μm filtrate was mixed 1:1 with 3.0M (NH4)2SO4 and the mixturewas applied to a Phenyl-sepharose FF (high sub) column (from GEHealthcare) equilibrated in 100 mM H3BO3, 10 mM MES/NaOH, 2 mM CaCl2),1.5M (NH4)2SO4, pH 6.0. After washing the column with the equilibrationbuffer, the protease was step-eluted with 100 mM H3BO3, 10 mM MES, 2 mMCaCl2), pH 6.0. The eluted peak (containing the protease activity) wascollected and applied to a Bacitracin agarose column (from Upfrontchromatography) equilibrated in 100 mM H3BO3, 10 mM MES, 2 mM CaCl2), pH6.0. After washing the column extensively with the equilibration buffer,the protease was eluted with 100 mM H3BO3, 10 mM MES, 2 mM CaCl2), 1MNaCl, pH 6.0 with 25% (v/v) 2-propanol. The elution peak (containing theprotease activity) was transferred to 20 mM MES, 2 mM CaCl2, pH 6.0 on aG25 sephadex column (from GE Healthcare). The G25 transferred peak wasthe purified preparation and was used for further experiments.

TABLE 1a Variants of SEQ ID NO 3 S114Q, S173P, S175P, F180Y K146T,S173P, S175P, F180Y K146N, S173P, S175P, F180Y K146W, S173P, S175P,F180Y K146F, S173P, S175P, F180Y K146A, S173P, S175P, F180Y I150A,S173P, S175P, F180Y I150N, S173P, S175P, F180Y S27K, I150N, S171N,S173P, G174R, S175P, F180Y, Q198E, T297P S27K, K146P, S148R, A151R,S171N, S173P, G174R, S175P, F180Y, Q198E, N199K, T297P S173P, S175P,N176Y, F180Y S173P, S175P, I178Y, F180Y S173P, S175P, I178F, F180YS173P, S175P, I178F, F180Y S173P, S175P, I178P, F180Y S173P, S175P,F180Y, L184F S173P, S175P, F180Y, L184F S27K, S171N, S173P, G174R,S175P, F180Y, L184F, Q198E, T297P S27K, I121V, S171N, S173P, G174R,S175P, F180Y, L184F, Q198E, T297P S27K, Q70N, G107N, I121V, E127Q,S173P, S175P, F180Y, L184F, Q198E, T297P S27K, S173P, G174K, S175P,F180Y, L184F, Q198E, N199K, T297P S27K, S173P, G174K, S175P, F180Y,L184F, Q198E, N199R, T297P S173P, S175P, F180Y, L184Y S27K, S171N,S173P, G174R, S175P, F180Y, L184Y, Q198E, T297P S27K, S173P, G174K,S175P, F180Y, L184Y, Q198E, N199K, T297P S27K, S173P, G174K, S175P,F180Y, L184Y, Q197K, Q198E, T297P S173P, S175P, F180Y, L184W S27K,S171N, S173P, G174R, S175P, F180Y, L184W, Q198E, T297P S173P, S175P,F180Y, L184D S173P, S175P, F180Y, R224D S173P, S175P, F180Y, R224G S27K,S171N, S173P, G174R, S175P, F180Y, Q198E, N199K, R224G, T297P S173P,S175P, F180Y, R224S S27K, I121V, S171N, S173P, G174R, S175P, F180Y,Q198E, R224S, T297P

TABLE 1b Variants of SEQ ID NO 3 S27K, S171D, S173P, G174R, S175P,G182A, L184F, Q198E, N199K, R224G, T297P S27K, S171D, S173P, G174R,S175P, G182A, L184F, Q198E, N199K, T297P S27K, S171N, S173P, G174R,S175P, F180Y, G182A, L184F, Q198E, N199K, R224G, T297P S27K, S171N,S173P, G174R, S175P, F180Y, G182A, L184F, Q198E, N199K, T297P S27K,V162T, S173P, G174K, S175P, F180Y, L184F, Q197K, Q198E, T297P S27K,V162T, S173P, G174K, S175P, F180Y, Q197K, Q198E, T297P

TABLE 1c Variants of SEQ ID NO 3 Q70F Q70A Q70N S111R S111E S111D S114AS144R A145E I150N I150S G182A L184F L184Y R224G Y240R

Example 2: Determination of Binding Constants of Inhibitor

Purified protease variants are pre-diluted to approximately 0.2 mg/ml indilution buffer. 40 μl diluted protease is then mixed with 40 μlinhibitor solution (Z-Gly-Ala-NHCH(CH₂C₆H₄pOH)C(OH)(SO₃Na)—H, wherein Zis benzyloxycarbonyl) or 40 μl 4-FPBA solution in the well of a 96 wellmicrotiter plate (Nunc F 96-MTP). For each protease variant 8concentrations of inhibitor solutions (600 μM, 200 μM, 67 μM, 22 μM, 7.4μM, 2.47 μM, 0.82 μM and 0 μM) and 8 concentrations of 4-FPBA (120 mM,40 mM, 13.3 mM, 4.44 mM, 1.48 mM, 0.494 mM, 0.164 mM and 0 mM) aretested. After 10 min mixing of protease and inhibitor at roomtemperature, 30 μl of the mixture is transferred to a microtiter plate(Nunc U96 PP 0.5 ml) with 270 μl Model B detergent in the wellsresulting in inhibitor concentrations of 0-30 μM and 4-FPBAconcentrations of 0-6000 μM. Protease, inhibitor and detergent is mixedfor 1 hour using magnetic bars for 1 hour at room temperature to reachequilibrium. Then 20 μl is transferred to a microtiter plate (Nunc F96-MTP). 100 μl substrate solution is added and after 5 sec mixingabsorbance at 405 nm is measured every 20 sec for 5 min on a SpectraMaxPlus reader. Slope from linear regression of initial increase inabsorbance at 405 nm is used for calculation of apparent bindingconstants.

TABLE 2 Experimental Conditions for determination of inhibitor bindingconstants Dilution buffer 0.01% Triton X-100 Detergent Model B Substratebuffer 100 mM Tris, pH 8.6 Substrate stock 100 mg/mlSuc-Ala-Ala-Pro-Phe-pNA (Bachem L-1400) in DMSO Substrate solution 7μl/ml substrate stock solution in substrate buffer InhibitorZ-Gly-Ala-NHCH(CH2C6H4pOH)C(OH)(SO3Na)-H, stock solution wherein Z isbenzyloxycarbonyl (414 mM) inhibitor solutions inhibitor stock dilutedto 600 μM, 200 μM, 67 μM, 22 μM, 7.4 μM, 2.47 μM, 0.82 μM and 0 μM withdilution buffer

TABLE 3 Composition of laundry liquid model B detergent Ingredient wt %(C10-C13)alkylbenzene sulfonic acid 7.2 sodium lauryl ether sulfate 10.6cocoa fatty acid 2.75 soy fatty acid 2.75 alcohol ethoxylate with 6.6 8mol EO sodium hydroxide 1.1 Ethanol 3 propane-1,2-diol 6 Glycerol 1.7Triethanolamine 3.3 sodium formiate 1 sodium citrate 2diethylenetriaminepentakis(methylene)pentakis(phosphonic acid), 0.5heptasodium salt copoly(acrylic acid/maleic acid), sodium salt 0.5deionized water 51

Calculation of Ki

It is assumed that protease and inhibitor react according to theequation:

E+I

EI

Where E is the protease, I is the inhibitor and EI is theprotease-inhibitor complex. It is furthermore assumed that equilibriumbetween protease, inhibitor and protease-inhibitor complex is reachedduring the 1 hour incubation in Model B, and that addition of substratesolution does not result in significant alteration of this equilibriumduring the measuring time used for linear regression. The concentrationof enzyme-inhibitor in the detergent solution is then given by:

$\lbrack{EI}\rbrack = \left( {{\left( {\left\lbrack E_{tot} \right\rbrack + \left\lbrack I_{tot} \right\rbrack + {K_{i}\text{-}{{sqrt}\left( {\left( {\left\lbrack E_{tot} \right\rbrack + \left\lbrack I_{tot} \right\rbrack + K_{i}} \right)^{\bigwedge}2\text{-}{{4^{*}\left\lbrack E_{tot} \right\rbrack}^{*}\left\lbrack I_{tot} \right\rbrack}} \right)}}} \right)\text{/}{2\mspace{76mu}\lbrack{EI}\rbrack}} = \frac{\left\lbrack E_{tot} \right\rbrack + \left\lbrack I_{tot} \right\rbrack + K_{i} - \sqrt{\begin{matrix}{\left( {\left\lbrack E_{tot} \right\rbrack + \left\lbrack I_{tot} \right\rbrack + K_{i}} \right)^{2} -} \\{4*\left\lbrack E_{tot} \right\rbrack*\left\lbrack I_{tot} \right\rbrack}\end{matrix}}}{2}} \right.$

where E_(tot) is the total protease concentration ([E_(tot)]=[E]+[EI]),I_(tot) is the total inhibitor concentration ([I_(tot)]=[I]+[EI]), andK_(i) is the equilibrium binding constant for the reaction. The measuredslopes V are given by:

$V = {{{V_{0}}^{*}\left( {1 - {\lbrack{EI}\rbrack {\text{/}\left\lbrack E_{tot} \right\rbrack}}} \right)} = {{V_{0}}^{*}\left( {{1 - {\left( {\left( {\left\lbrack E_{tot} \right\rbrack + \left\lbrack I_{tot} \right\rbrack + {K_{i}\text{-}{{sqrt}\left( {\left( {\left\lbrack E_{tot} \right\rbrack + \left\lbrack I_{tot} \right\rbrack + K_{i}} \right)^{\bigwedge}2\text{-}{{4^{*}\left\lbrack E_{tot} \right\rbrack}^{*}\left\lbrack I_{tot} \right\rbrack}} \right)}}} \right)\text{/}2{\text{/}\left\lbrack E_{tot} \right\rbrack}} \right)\mspace{76mu} V}} = {{V_{0}*\left( {1 - \frac{\lbrack{EI}\rbrack}{\left\lbrack E_{tot} \right\rbrack}} \right)} = {V_{0}*\left( {1 - \frac{\begin{matrix}{\left\lbrack E_{tot} \right\rbrack + \left\lbrack I_{tot} \right\rbrack + K_{i} -} \\\sqrt{\begin{matrix}{\left( {\left\lbrack E_{tot} \right\rbrack + \left\lbrack I_{tot} \right\rbrack + K_{i}} \right)^{2} -} \\{4*\left\lbrack E_{tot} \right\rbrack*\left\lbrack I_{tot} \right\rbrack}\end{matrix}}\end{matrix}}{2*\left\lbrack E_{tot} \right\rbrack}} \right)}}} \right.}}$

where V₀ is the slope without inhibitor added. To calculate apparentbinding constant K_(i) least square fitting of measured slopes atvarious inhibitor concentrations to this equation is performed withK_(i) and V₀ as variables.

The K_(i) for the parent protease and for each variant was calculated asdescribed above

Having the K_(i) for each variant, it is possible to calculate theeffect of a single mutation, if data are available for at least twovariants that differ only by that mutation. In this case, the relativeK_(i) for the mutation is calculated:

${{Relative}\mspace{14mu} {Ki}_{mutation}} = \frac{{Ki}_{{variant}\mspace{14mu} {with}\mspace{14mu} {mutation}}}{{Ki}_{{variant}\mspace{14mu} {without}\mspace{14mu} {mutation}}}$

A lower K_(i) is desirable, as a satisfactory inhibition can be obtainedat a lower inhibitor concentration, so beneficial mutations have arelative K_(i)<1.0The following table show such relative Ki_(mutation). Each row show:

-   -   a mutation (column 1: “Mutation”)    -   a protease variant (relative to SEQ ID NO: 2) that comprise the        mutation from column 1 (column 2: “Variant with the mutation”)    -   another protease variant (relative to SEQ ID NO: 2), that        differs from the one in column 2 only by not having the mutation        in column 1 (column 3: “Variant without the mutation”)    -   Relative Ki_(mutation) for the mutation in column 1, in the        context of the variant in column 3, measured in Model B        detergent (column 4: “inhibitor binding in Model B”)    -   Relative Ki_(mutation) for the mutation in column 1, in the        context of the variant in column 3, measured in Tris buffer at        pH 8 (column 5: “inhibitor binding in Tris buffer pH 8”)

TABLE 4 Inhibitor-binding relative Ki_(mutation) inhibitor inhibitorbinding in Variant without the binding in Tris buffer Mutation Variantwith the mutation mutation Model B pH 8 S114Q S114Q, S173P, S175P, F180YS173P, S175P, F180Y 0.63 0.34 K146T K146T, S173P, S175P, F180Y S173P,S175P, F180Y 0.08 0.06 K146N K146N, S173P, S175P, F180Y S173P, S175P,F180Y 0.93 0.91 K146W K146W, S173P, S175P, F180Y S173P, S175P, F180Y0.23 0.16 K146F K146F, S173P, S175P, F180Y S173P, S175P, F180Y 0.25 0.19K146A K146A, S173P, S175P, F180Y S173P, S175P, F180Y 0.34 0.29 I150AI150A, S173P, S175P, F180Y S173P, S175P, F180Y 0.50 0.40 I150N I150N,S173P, S175P, F180Y S173P, S175P, F180Y 0.35 0.21 I150N S27K, I150N,S171N, S173P, S27K, S171N, S173P, 0.24 ND G174R, S175P, F180Y, Q198,G174R, S175P, F180Y, ET297P Q198E, T297P A151R S27K, K146P, S148R,A151R, S27K, K146P, S148R, S171N, 0.75 ND S171N, S173P, G174R, S175P,S173P, G174R, S175P, F180Y, F180Y, Q198E, N199K, T297P Q198E, N199K,T297P N176Y S173P, S175P, N176Y, F180Y S173P, S175P, F180Y 0.81 ND I178YS173P, S175P, I178Y, F180Y S173P, S175P, F180Y 0.53 ND I178F S173P,S175P, I178F, F180Y S173P, S175P, F180Y 0.50 ND I178F S173P, S175P,I178F, F180Y S173P, S175P, F180Y 0.49 ND I178P S173P, S175P, I178P,F180Y S173P, S175P, F180Y 0.51 ND L184F S173P, S175P, F180Y, L184FS173P, S175P, F180Y 0.31 ND L184F S173P, S175P, F180Y, L184F S173P,S175P, F180Y 0.27 ND L184F S27K, S171N, S173P, G174R, S27K, S171N,S173P, G174R, 0.18 ND S175P, F180Y, L184F, Q198E, S175P, F180Y, Q198E,T297P T297P L184F S27K, I121V, S171N, S173P, G174R, S27K, I121V, S171N,S173P, 0.30 ND S175P, F180Y, L184F, G174R, S175P, F180Y, Q198E, T297PQ198E, T297P L184F S27K, Q70N, G107N, I121V, E127Q, S27K, Q70N, G107N,I121V, 0.37 ND S173P, S175P, F180Y, E127Q, S173P, S175P, L184F, Q198E,T297P F180Y, Q198E, T297P L184F S27K, S173P, G174K, S175P, S27K, S173P,G174K, S175P, 0.36 ND F180Y, L184F, Q198E, N199K, F180Y, Q198E, N199K,T297P T297P L184F S27K, S173P, G174K, S175P, S27K, S173P, G174K, S175P,0.22 ND F180Y, L184F, Q198E, N199R, F180Y, Q198E, N199R, T297P T297PL184Y S173P, S175P, F180Y, L184Y S173P, S175P, F180Y 0.24 ND L184Y S27K,S171N, S173P, G174R, S27K, S171N, S173P, 0.18 ND S175P, F180Y, L184Y,Q198E, G174R, S175P, F180Y, T297P Q198E, T297P L184Y S27K, S173P, G174K,S175P, S27K, S173P, G174K, 0.21 ND F180Y, L184Y, Q198E, N199K, S175P,F180Y, Q198E, T297P N199K, T297P L184Y S27K, S173P, G174K, S175P, S27K,S173P, G174K, 0.32 ND F180Y, L184Y, Q197K, Q198E, S175P, F180Y, Q197K,T297P Q198E, T297P L184W S173P, S175P, F180Y, L184W S173P, S175P, F180Y0.50 ND L184W S27K, S171N, S173P, G174R, S27K, S171N, S173P, 0.57 NDS175P, F180Y, L184W, Q198E, G174R, S175P, F180Y, T297P Q198E, T297PL184D S173P, S175P, F180Y, L184D S173P, S175P, F180Y 0.37 ND R224DS173P, S175P, F180Y, R224D S173P, S175P, F180Y 0.36 ND R224G S173P,S175P, F180Y, R224G S173P, S175P, F180Y 0.47 ND R224G S27K, S171N,S173P, G174R, S27K, S171N, S173P, 0.74 ND S175P, F180Y, Q198E, N199K,G174R, S175P, F180Y, R224G, T297P Q198E, N199K, T297P R224S S173P,S175P, F180Y, R224S S173P, S175P, F180Y 0.50 ND R224S S27K, I121V,S171N, S173P, G174R, S27K, I121V, S171N, 0.56 ND S175P, F180Y, Q198E,S173P, G174R, S175P, F180Y, R224S, T297P Q198E, T297P

TABLE 4b Inhibitor-binding relative Ki_(mutation). Variant without theinhibitor binding in Mutation Variant with the mutation mutation Model BR224G S27K, S171D, S173P, G174R, S27K, S171D, S173P, G174R, 0.49 S175P,G182A, L184F, Q198E, S175P, G182A, L184F, Q198E, N199K, R224G, T297PN199K, T297P R224G S27K, S171N, S173P, G174R, S27K, S171N, S173P, G174R,0.69 S175P, F180Y, G182A, L184F, S175P, F180Y, G182A, L184F, Q198E,N199K, R224G, T297P Q198E, N199K, T297P L184F S27K, V162T, S173P, G174K,S27K, V162T, S173P, G174K, 0.17 S175P, F180Y, L184F, Q197K, S175P,F180Y, Q197K, Q198E, Q198E, T297P T297P

Example 4: Determination of Binding Constants in Model B Detergent

K_(i) were calculated as shown in Example 3. The experimental conditionsare shown in table 5. The Improvement Factor (IF) or Relative

${Ki}_{mutation} = \frac{{Ki}_{{variant}\mspace{14mu} {with}\mspace{14mu} {mutation}}}{{Ki}_{{variant}\mspace{14mu} {without}\mspace{14mu} {mutation}}}$

for the inhibitors NNIC and 4-FPBA are shown in tables 6 and 7.

TABLE 5 Experimental Conditions Dilution buffer 0.01% Triton X-100Detergent Model B Substrate buffer 100 mM Tris, pH 8.6 Substrate stock100 mg/ml Suc-Ala-Ala-Pro-Phe-pND (Bachem L-1400) in DMSO Substratesolution 7 μl/ml substrate stock solution in substrate buffer InhibitorZ-Gly-Ala-NHCH(CH2C6H4pOH)C(OH)(SO3ND)-H, NNIC stock wherein Z isbenzyloxycarbonyl (414 mM) solution NNIC solutions NNIC stock diluted to600 μM, 200 μM, 67 μM, 22 μM, 7.4 μM, 2.47 μM, 0.82 μM and 0 μM withdilution buffer 4-FPBA stock 4-FPBA (1801 mM) solution 4-FPBA solutions4-FPBA stock solution diluted to 120 mM, 40 mM, 13.3 mM, 4.44 mM, 1.48mM, 0.494 mM, 0.164 mM and 0 mM with dilution buffer

TABLE 6 Inhibitor (NNIC) binding constant improvement factors in Model Brelative to TY145 backbone Mutation IF Y240R 0.84 S111R 0.90 Q70F 0.12Q70A 0.39 Q70N 0.30 S114A 0.47 I150N 0.27 I150S 0.20 G182A 0.54 L184F0.44 L184Y 0.22 R224G 0.60 TY145 1.00

TABLE 7 Inhibitor (4-FPBA) binding constant improvement factors in ModelB relative to TY145 backbone Mutation IF S111E 0.81 S111D 0.65 A145E0.97 S144R 0.49 Q70N 0.28 S114A 0.71 L184F 0.72 TY145 1.00

1. A protease variant comprises one or more substitutions selected fromthe group consisting of Q70F, Q70A, Q70N, S111R, S111E, S111D, S114A,S114Q, S144R, A145E, K146T, K146N, K146W, K146F, K146A, I150A, I150N,I150N, I150S,A151R, N176Y, I178Y, I178F, I178P, G182A, L184F, L184Y,L184F, L184Y, L184W, L184D, R224D, R224G, R224S, and Y240R, wherein thepositions corresponds to the positions of SEQ ID NO: 3, wherein thevariant has a sequence identity to SEQ ID NO: 3 of at least 70% andwherein the variant has protease activity.
 2. A protease variant ofclaim 1, which has reduced Ki compared to the parent or compared to theprotease with SEQ ID NO:
 3. 3. A protease variant of claim 1, whereinthe variant is selected from the group consisting of: a) a polypeptidehaving at least 60% sequence identity to the mature polypeptide of SEQID NO: 2; b) a polypeptide encoded by a polynucleotide that hybridizesunder medium, or high stringency conditions with (i) the maturepolypeptide coding sequence of SEQ ID NO: 1, (ii) a sequence encodingthe mature polypeptide of SEQ ID NO: 2, or (iii) the full-lengthcomplement of (i) or (ii); c) a polypeptide encoded by a polynucleotidehaving at least 75% identity to the mature polypeptide coding sequenceof SEQ ID NO: 1 or a sequence encoding the mature polypeptide of SEQ IDNO: 2; and d) a fragment of the mature polypeptide of SEQ ID NO: 2,which has protease activity e) a protease variant having at least 60%sequence identity to the mature polypeptide of SEQ ID NO: 2, andcomprising an alteration selected from the group consisting ofS114Q,S173P,S175P,F180Y K146T,S173P,S175P,F180Y K146N,S173P,S175P,F180YK146W,S173P,S175P,F180Y K146F,S173P,S175P,F180Y K146A,S173P,S175P,F180YI150A,S173P,S175P,F180Y I150N,S173P,S175P,F180YS27K,I150N,S171N,S173P,G174R,S175P,F180Y,Q198E,T297PS27K,K146P,S148R,A151R,S171N,S173P,G174R,S175P,F180Y,Q198E,N199K,T297PS173P,S175P,N176Y,F180Y S173P,S175P,I178Y,F180Y S173P,S175P,I178F,F180YS173P,S175P,I178F,F180Y S173P,S175P,I178P,F180Y S173P,S175P,F180Y,L184FS173P,S175P,F180Y,L184FS27K,S171N,S173P,G174R,S175P,F180Y,L184F,Q198E,T297PS27K,I121V,S171N,S173P,G174R,S175P,F180Y,L184F,Q198E,T297PS27K,Q70N,G107N,I121V,E127Q,S173P,S175P,F180Y,L184F,Q198E,T297PS27K,S173P,G174K,S175P,F180Y,L184F,Q198E,N199K,T297PS27K,S173P,G174K,S175P,F180Y,L184F,Q198E,N199R,T297PS173P,S175P,F180Y,L184YS27K,S171N,S173P,G174R,S175P,F180Y,L184Y,Q198E,T297PS27K,S173P,G174K,S175P,F180Y,L184Y,Q198E,N199K,T297PS27K,S173P,G174K,S175P,F180Y,L184Y,Q197K,Q198E,T297PS173P,S175P,F180Y,L184WS27K,S171N,S173P,G174R,S175P,F180Y,L184W,Q198E,T297PS173P,S175P,F180Y,L184D S173P,S175P,F180Y,R224D S173P,S175P,F180Y,R224GS27K,S171N,S173P,G174R,S175P,F180Y,Q198E,N199K,R224G,T297PS173P,S175P,F180Y,R224SS27K,I121V,S171N,S173P,G174R,S175P,F180Y,Q198E,R224S,T297PS27K,S171D,S173P,G174R,S175P,G182A,L184F,Q198E,N199K,R224G,T297PS27K,S171D,S173P,G174R,S175P,G182A,L184F,Q198E,N199K,T297PS27K,S171N,S173P,G174R,S175P,F180Y,G182A,L184F,Q198E,N199K,R224G,T297PS27K,S171N,S173P,G174R,S175P,F180Y,G182A,L184F,Q198E,N199K,T297PS27K,V162T,S173P,G174K,S175P,F180Y,L184F,Q197K,Q198E,T297PS27K,V162T,S173P,G174K,S175P,F180Y,Q197K,Q198E,T297P Q70F Q70A Q70NS111R S111E S111D S114A S144R A145E I150N I150S G182A L184F L184Y R224Gand Y240R
 4. A protease variant of claim 1, wherein the variant has atleast 70%, at least 80%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99 sequence identity to the maturepolypeptide of SEQ ID NO:
 3. 5. A protease variant of claim 1, whereinthe total number of alterations compared to SEQ ID NO: 3 is 1-20, e.g.1-10 and 1-5, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or15 alterations.
 6. A detergent composition comprising a protease variantaccording to claim
 1. 7. The detergent composition of claim 6, furthercomprising one or more detergent components.
 8. The detergentcomposition according to claim 6, further comprising one or moreadditioNDI enzymes selected from the group comprising of proteases,amylases, lipases, cutiNDses, cellulases, endoglucaNDses,xyloglucaNDses, pectiNDses, pectin lyases, xanthaNDses, peroxidaes,haloperoxygeNDses, catalases and manNDNDses, or any mixture thereof. 9.The detergent composition according to claim 6 in form of a bar, ahomogenous tablet, a tablet having two or more layers, a pouch havingone or more compartments, a regular or compact powder, a granule, apaste, a gel, or a regular, compact or concentrated liquid. 10.(canceled)
 11. A method for obtaining a protease variant, comprisingintroducing into a parent protease one or more of the followingsubstitutions: Q70F, Q70A, Q70N, S111R, S111E, S111D, S114A, S114Q,S144R, A145E, K146T, K146N, K146W, K146F, K146A, I150A, I150N, I150N,I150S,A151R, N176Y, I178Y, I178F, I178P, G182A, L184F, L184Y, L184F,L184Y, L184W, L184D, R224D, R224G, R224S, and Y240R of SEQ ID NO 3,wherein the variant has an amino acid sequence which is at least 60%identical to SEQ ID NO: 3, and recovering the variant.
 12. The method ofclaim 11, wherein the variant comprises two, three, four or fivesubstitutions corresponding to positions 114, 146, 150, 151, 176, 178,184 or 224 of the mature polypeptide of SEQ ID NO:
 3. 13. The methodaccording to claim 11, wherein the protease variant has at least 60%,such as at least 70%, at least 80%, at least 90%, at least 95%, at least96%, at least 97%, at least 98%, or at least 99%, sequence identity tothe mature polypeptide of SEQ ID NO:
 3. 14. The method according toclaim 11, wherein the parent protease selected from the group consistsof: a) a polypeptide having at least 60% sequence identity to the maturepolypeptide of SEQ ID NO: 2; b) a polypeptide encoded by apolynucleotide that hybridizes under medium or high stringencyconditions with (i) the mature polypeptide coding sequence of SEQ ID NO:1, (ii) a sequence encoding the mature polypeptide of SEQ ID NO: 2, or(iii) the full-length complement of (i) or (ii); c) a polypeptideencoded by a polynucleotide having at least 60% identity to the maturepolypeptide coding sequence of SEQ ID NO: 1 or a sequence encoding themature polypeptide of SEQ ID NO: 2; and d) a fragment of the maturepolypeptide of SEQ ID NO: 2, which has protease activity.
 15. The methodaccording to claim 11, wherein the parent protease has at least 75%,such as at least 80%, at least 85%, at least 90%, at least 95%, at least96%, at least 97%, at least 98%, at least 99%, sequence identity to SEQID NO: 3.